Genetically modified pigs for xenotransplantation of vascularized xenografts and derivatives thereof

ABSTRACT

The present invention provides certain donor animals, tissues and cells that are particularly useful for xenotransplantation therapies. In particular, the invention includes porcine animals, as well as tissue and cells derived from these, which lack any expression of functional alpha 1,3 galactosyltransferase (aGT) and express one or more additional transgenes which make these animals suitable donors for xenotransplantation of vascularized xenografts and derivatives thereof. Methods of treatment and using organs, tissues and cells derived from such animals are also provided.

This application claims priority to U.S. provisional patent application61/442,504, filed on Feb. 14, 2011.

FIELD OF THE INVENTION

The present invention provides certain donor animals, tissues and cellsthat are particularly useful for xenotransplantation therapies. Inparticular, the invention includes porcine animals, as well as tissueand cells derived from these, which lack any expression of functionalalpha 1,3 galactosyltransferase (αGT) and express one or more additionaltransgenes which make these animals suitable donors forxenotransplantation of vascularized xenografts and derivatives thereof.Methods of treatment and using organs, tissues and cells derived fromsuch animals are also provided.

BACKGROUND OF THE INVENTION

There is a critical shortage of human organs for the purposes of organtransplantion. In the United States alone approximately 110,000 patientsare on waiting lists to receive organs, and yet only 30,000 organs willbecome available from deceased donors. Almost 20 patients die each day(7,000 per year) waiting for an organ (Cooper and Ayares, 2010International Journal of Surgery, In Press,doi:10.1016/j.ijsu.2010.11.002). The supply of human organs and tissuesfor use in allotransplantion will never fully meet the population'sneed. A new source of donor materials is urgently needed.

Xenotransplantation

Xenotransplantation (transplant of organs, tissues and cells from adonor of a different species) could effectively address the shortage ofhuman donor material. Xenotransplants are also advantageously (i)supplied on a predictable, non-emergency basis; (ii) produced in acontrolled environment; and (iii) available for characterization andstudy prior to transplant.

Depending on the relationship between donor and recipient species, thexenotransplant can be described as concordant or discordant. Concordantspecies are phylogenetically closely related species (e.g., mouse torat). Discordant species are not closely related (e.g., pig to human).Pigs have been the focus of most research in the xenotransplanationarea, since the pig shares many anatomical and physiologicalcharacteristics with human. Pigs also have relatively short gestationperiods, can be bred in pathogen-free environments and may not presentthe same ethical issues associated with animals not commonly used asfood sources (e.g., primates).

Scientific knowledge and expertise in the field of pig-to-primatexenotransplantation has grown rapidly over the last decade, resulting inthe considerably prolonged survival of primate recipients of lifesavingporcine xenografts. (Cozzi et al., Xenotransplantation, 16:203-214.2009). Recently, significant achievements have been reported in thefield of organ xenotransplantation. For a review of organxenotransplantation results, see Ekser et al., 2009, TransplantImmunology June; 21(2): 87-92

Genetic Modification

While advantageous in many ways, xenotransplantation also creates a morecomplex immunological scenario than allotransplantation. As such,considerable effort has been directed at addressing the immune barrierthrough genetic modification (van der Windt et al., Xenotransplantation.2007 Jul.; 14(4):288-97, Cowan and D'Apice, Curr Opin Organ Transplant.2008 Apr.; 13(2):178-83).

Xenograft rejection can be divided into three phases: hyperacuterejection, acute humoral xenograft rejection, and T cell-mediatedcellular rejection. Hyperacute rejection (HAR) is a very rapid eventthat results in irreversible graft damage and loss within minutes tohours following graft reperfusion. It is triggered by the presence ofxenoreactive natural antibodies present within the recipient at the timeof transplantation. Humans have a naturally occurring antibody to thealpha 1,3-galactose (Gal) epitope found on pig cells. This antibody isproduced in high quantity and, it is now believed, is the principlemediator of HAR. (Sandrin et al., Proc Natl Acad Sci 1993 Dec. 1;90(23):11391-5, 1993; review by Sandrin and McKenzie, Immunol Rev. 1994October; 141:169-90).

Initial efforts to genetically modify pigs have focused on removing thealpha 1,3-galactose (Gal) epitope from pig cells. In 2003, Phelps et al.(Science, 2003, 299:411-414) reported the production of the first livepigs lacking any functional expression of αGT (GTKO), which representeda major breakthrough in xenotransplantation (see also PCT publicationNo. WO 04/028243 to Revivicor, Inc. and PCT Publication No. WO 04/016742to Immerge Biotherapeutics, Inc.). Subsequent studies have shown thatorgan grafts from GTKO pigs do not undergo HAR (Kuwaki et al., Nat Med.2005 Jan.; 11(1):29-31, Yamada et al., Nat Med. 2005 Jan.; 11(1):32-4).Expression of complement regulators in xenotransplant tissue has beensuggested as a different strategy to combat HAR (Squinto, Curr OpinBiotechnol. 1996 Dec.; 7(6):641-5). European patent 0495852 to Imutransuggests associating xenograft tissues with recipient complementrestriction factors to reduce complement activation in the recipient(see also Diamond, et al., Transpl Immunol. 1995 Dec.; 3(4):305-12).Transgenic pigs expressing human DAF (hDAF) and/or human CD59 (hCD59)have been reported (Byrne et al., Transplant Proc., 1996 Apr.;28(2):758).

CD46 has been expressed in pig cells using a minigene that was optimizedfor high ubiquitous expression and kidneys from these CD46 transgenicpigs were protected from hyperacute rejection in a primatexenotransplantation model (Loveland et al., Xenotransplantation, 2004,11:171:183). Transgenic pigs with the combination of GTKO and expressionof CD46 were recently tested in a heterotopic heart model(pig-to-baboon) and provided prolonged survival and function ofxenograft hearts for up to 8 months without any evidence of immunerejection (Mohiuddin et al., Abstract TTS-1383. Transplantation 2010; 90(suppl): 325; Mohiuddin et al. (2011), online publication AmericanJournal of Transplantation). However, the primates were placed on a ATG,anti-CD154 and MMF-based immunosuppressive regimen, which prolongedGTKO.hCD46Tg graft survival for up to 236 days (n=9, median survival 71days and mean survival 94 days). It is not possible for this type ofimmunosuppressive regimen to be used in humans. Ekser et al.(Transplantation 2010 Sep. 15; 90(5):483-93) reported hepatic functionin baboons after transplant of livers fromalpha-1,3-galactosyltransferase gene-knockout pigs transgenic for CD46.The recipient baboons died or were euthanized after 4 to 7 days but waslimited by the rapid development of a profound thrombocytopenia.

Even where HAR is avoided, the xenograft undergoes a delayed form ofrejection, acute humoral or acute vascular xenograft rejection(AHXR/AVXR)— also referred to as delayed xenograft rejection (DXR)(Shimizu et al 2008 Am J Pathol. 2008 June; 172(6):1471-81). It isgenerally thought to be initiated by xeno-reactive antibodies, includingnon-Gal antibodies and subsequent activation of the graft endothelium,the complement and the coagulation systems (Miyagawa et al.Xenotransplantation, 2010, 1: 11-25). Although the threats presented bythe humoral response are critical with regard to the survival andfunction of vascularized grafts, the risk of graft damage by cellularmechanisms is also important. T-cell mediated acute responses play animportant role in xenotransplant rejection. Of several T cellcostimulatory pathways identified to date, the most prominent is theCD28 pathway and the related cytoxic T-lymphocyte associated protein(CTLA4) pathway.

To date, much of the research on CTLA4-Ig as an immunosuppressive agenthas focused on administering soluble forms of CTLA4-Ig to a patient (seeU.S. Pat. No. 7,304,033; PCT Publication No. WO 99/57266; and Lui et al.J Immunol Methods 2003 277:171-183). To reduce the overallimmunosuppressive burden on a patient, transgenic expression of such aprotein has been suggested. Transgenic mice expressing CTLA4-Ig havebeen developed (Ronchese et al. J Exp Med (1994) 179:809; Lane et al. JExp Med. (1994) Mar. 1; 179(3):819; Sutherland et al. Transplantation.2000 69(9):1806-12). In addition, PCT Publication No. WO 01/30966 toAlexion Pharmaceuticals, Inc. and PCT Publication No. WO 07/035,213 toRevivicor discloses transgenic pigs expressing only the CTLA4-Igtransgene (see also Phelps et al., Xenotransplantation, 16(6):477-485.2009). Pigs expressing CTLA4-Ig in brain tissue were produced, but highplasma expression was shown to cause negative effects (Martin, et al.(2005) Transg. Rsch. 14:373-84). There remains doubt as to whether longterm expression of immunosuppressive transgenes in ungulates raisessafety concerns either for the ungulate or for the recipient of anytissues from such an animal.

In addition to the cellular and humoral immune responses, a significantchallenge associated with xenotransplantation is coagulationdysregulation and thrombotic microangiopathy in the vasculature of thegraft (Ekser et al, 2009 Jun.; 21 (2):87-92). This phenomenon came tolight when HAR was prevented by removal of the Gal epitope and/ortransgenic expression of complement inhibitors, and classical AHXR wasalso prevented with high levels of immunosuppression. This intravascularcoagulation is triggered by either antibody/cell-mediated damage of theendothelium or by coagulation factor incompatibilities between thediscordant species (pig and non-human primate), which leads toendothelial activation. In a transplant setting, the endothelium of adonor vascularized graft is where donor antigens come into contact witha recipient's (or host's) bloodstream, leading to an antibody mediatedimmune response, and potential rejection of the graft. Depending on therelatedness of the donor and the recipient, various types of immunerejection can occur. (see, for example, Fundamental Immunology, Ed.William E. Paul, Lippincott Williams & Wilkins; Sixth edition (May 22,2008), Chapter 44)

Once the endothelium is activated, it changes from its anticoagulantstate to a procoagulant state by up regulation of von Willebrand factorand production of tissue factor leading to thrombus formation,hemorrhage, and rejection of the graft (Mohiuddin, 2007, PLoS Medicine,Vol 4(3) p. 0429-0434). Until this intravascular coagulation issue isaddressed, obtaining long-term survival of a vascularized xenograft willremain a formidable challenge.

The addition of an anticoagulant transgene has been suggested to preventcoagulation responses to xenografts (reviewed by Cowan,Xenotransplantation, 2007; 14:7-12), yet to date, very few transgenicpigs expressing anticoagulants have been produced and none have beentested in vivo in xenotransplantation models. Significant health andviability issues in pigs produced with an anticoagulant phenotype hasled to a low rate of production of viable transgenic animals. In mice,expression of the anticoagulant CD39 driven by an murine H2-K (MHC classI) promoter, led to impaired platelet aggregation and prolonged bleedingtimes (see Dwyer et al. (2004) J Clin Invest 113: 1440-46). Anothergroup attempted to produce TFPI transgenic pigs in combination with aDAF transgene driven by the pCMVIE constitutive promoter enhancer vianuclear transfer (Lee et al 2010 Reprod Domest Anim. 2010 Jul. 4 epubahead of print PMID: 20626677). Only one viable transgenic piglet wasobtained from this effort and it was shown to express DAF and TFPI inheart, liver and ear cells examined.

Multi-transgenic pigs have also been generated which contain hCD59, DAFand TM transgenes, with the anticoagulant TM driven by a CMV promoter(Petersen et al., Xenotransplantation 2009:16:486-495), however therewas considerable variability in TM expression level in these pigs.Further, Dwyer et al. (Transplantation Reviews 21 (2007) 54-63), brieflyreported the generation of CD39 transgenic pigs. U.S. Pat. No. 7,378,569discloses transgenic pigs carrying two transgenes, one encoding a humandecay accelerating factor (hDAF) and the other encoding a human hemeoxygenase-1 (hHO)-1, which are useful for providing cells, tissues ororgans therefrom for xenotransplantation. Ayares (2009Xenotransplantation 16(5) 373) discusses further genetic modification,building on the GTKO genetic background, has been initiated by a numberof groups to address issues such as induced antibody responses tonon-Gal antigens, thrombosis, and cell-mediated immune responses.

Although xenotransplantation of organs, particularly from porcinedonors, is an appealing alternative to the use of allografts because ofthe limited supply and quality of human donor materials, major obstaclesremain. Both immediate and delayed immune responses require potentiallytoxic cocktails of immunosuppressant therapies, and even then,endothelial activation and subsequent coagulation dysregulation andthrombosis in the graft can cause graft failure. The production ofgenetically modified animals to address certain immune responses hasbeen suggested; however, this requires the coordinated elimination andappropriate expression of multiple transgenes that are capable ofaddressing each immune response without significantly curtailing theoverall health and viability of the pig. Thus, there remains a need forimproved animals and tissues suitable for xenotransplantation therapies.In particular, there remains a need for improved donor animals, organsand tissues for use in xenotransplantation without requiring significantor long term immunosuppressive or anticoagulant therapies.

It is an object of the present invention to provide genetically modifiedporcine animals for xenotransplantation of vascularized xenografts andderivatives thereof.

It is another object of the present invention to provide vascularizedxenografts from genetically modified pocine animals which express of oneor more immunosuppressant and/or anticoagulant transgenes and lackexpression of alpha-1,3-galactosyltransferase.

It is a further object of the present invention to provide geneticallymodified porcine animals which express of one or more immunosuppressantand/or anticoagulant transgenes spherically in the endothelium.

SUMMARY OF THE INVENTION

The present invention provides genetically modified porcine animals,organs, tissues and cells thereof that are particularly useful forxenotransplantation of vascularized xenografts and derivatives thereof.Vascularized xenografts can include any organ, tissue, cell orcombination thereof, that contains porcine blood vessels and/or isderived from an organ, tissue or cell therefrom. The geneticallymodified donor animals serve as a source of organs, tissues and cellsthat overcome significant humoral (HAR and AHXR/AVXR/DXR) and cellularimmune responses (ACXR), making them particular useful forxenotransplantation. Specifically, the genetically modified donoranimals have transgenes specifically expressed in the vascularendothelium.

Since the vascular endothelium is the site of first contact between therecipient's bloodstream and a donor graft, and the site of the initialimmune activation and response; modification of the pig's endotheliumwill reduce graft damage and rejection due to the consumptivecoagulopathy (also known as disseminated intravascular coagulation(DIC)), and thrombotic microangiopathy currently observed followingdiscordant xenotransplantation. Such genetically modified donor animalscan serve as a source of vascularized xenografts (as well as the organs,tissues and cells derived therefrom), making them particular useful forxenotransplantation, using a clinically relevant immunosuppressantregimen.

The viable, genetically modified porcine animals of the presentinvention are characterized by globally reduced immune reactivity (i.e.,due to the lack of expression of functional alpha 1,3 galactosyltransferase (αGT)) as well as the expression of transgenes critical toovercome transplant rejection, selected from the group includinganticoagulants, immunomodulators and cytoprotectants. Prior to thepresent invention, it was unknown whether these types of transgenes,which can cause the animal to be immuno-compromised and hemophilic,could be expressed in a single animal that would be able to be asuitable transplantation donor because it was expected that the animals'viability would be severely curtailed. The present inventors have foundthat such donor animals, tissues and cells can be obtained, inparticular when globally reduce immune reactivity due to lack ofexpression of functional alpha 1,3 galactosyltransferase (GTKO) iscombined with endothelial specific expression of certain transgenes.

Further embodiments of the present invention include the addition of animmunomodulator transgene that is specifically expressed in theendothelium. The immunomodulator can be an immunosuppressor molecule,such as CTLA4, in particular, CTLA4-Ig. The local expression of animmunomodulator allows for the ultimate use of a clinically relevantimmunosuppressant regimen in the human following xenotransplantation ofthe organ, tissue or cell.

In one embodiment of the present invention, GTKO porcine animals,organs, tissues and cells are provided that specifically express atleast one transgene in endothelium.

In a particular embodiment, the transgene specifically expressed inendothelium is at least one anticoagulant. In another particularembodiment, the transgene specifically expressed in endothelium is atleast one immunomodulator. In specific embodiment, the transgenespecifically expressed in endothelium is at least one immunosuppressant.In a further particular embodiment, the transgene specifically expressedin endothelium is at least one cytoprotective transgene.

In another embodiment of the present invention, GTKO animals, tissuesand cells are provided that specifically express multiple transgenes inendothelium. In a particular embodiment, the multiple transgenes areselected from the group that includes anticoagulants, immunomodulatorsand cytoprotective transgenes.

In a particular embodiment, GTKO animals, tissues and cells are providedthat specifically express at least two transgenes in endothelium. In aspecific embodiment, the at least two transgenes are bothanticoagulants.

In a particular embodiment, GTKO animals, tissues and cells are providedspecifically express at least three transgenes in endothelium. In aspecific embodiment, the at least three transgenes include twoanticoagulant transgenes and an immunosuppressant transgene.

In a further specific embodiment, GTKO animals, tissues and cells areprovided that lack any expression of functional alpha 1,3galactosyltransferase (GTKO) and that specifically expressthrombomodulin and EPCR (Endothelial Protein C Receptor) in endothelium.

In a further embodiment of the present invention, porcine animals,tissues and cells are provided that lack any expression of functionalalpha 1,3 galactosyltransferase (GTKO) and that express at least onefirst transgene and at least one second transgene, wherein the secondtransgene is specifically expressed in endothelium.

In one embodiment, the at least one first transgene is animmunomodulator. In a particular embodiment, the at least one firsttransgene is a compliment inhibitor.

In another embodiment, the at least one first transgene is a complimentinhibitor and the at least one second transgene specifically expressedin endothelium is selected from the group that includes (i) ananticoagulant; (ii) an immunosuppressive; and (iii) a cytoprotectant.

In one embodiment, porcine animals, tissues and cells are provided thatlack any expression of functional alpha 1,3 galactosyltransferase (GTKO)and expresses at least one compliment inhibitor and at least oneadditional transgene selected from the group consisting ofanticoagulants, immunosuppressants and cytoprotectants.

In a specific embodiment, porcine animals, tissues and cells are providethat lack any expression of functional alpha 1,3 galactosyltransferase(GTKO) and expresses at least one compliment inhibitor and at least oneanticoagulant. In a particular embodiment, the compliment inhibitor isCD46 and the at least one anticoagulant is selected from the group thatconsists of TFPI, CD39, hirudin, thrombomodulin and EPCR. In aparticular embodiment, the at least one compliment inhibitor is CD46 andthe at least one anticoagulant is thrombomodulin. In a furtherparticular embodiment, the at least one compliment inhibitor is CD46 andthe at least one additional transgene is an immunosuppressant, e.g.,CTLA4.

In a specific embodiment, porcine animals, tissues and cells areprovided that lack any expression of functional alpha 1,3galactosyltransferase (GTKO) and further express at least one complimentinhibitor, at least one anticoagulant and at least oneimmunosuppressant. Optionally, the porcine animals, tissues and cellsalso express at least one cytoprotective transgene.

In one embodiment, the transgene is specifically expressed inendothelium. In a particular embodiment, the transgene is specificallyexpressed in endothelial cells. In a specific embodiment, the transgeneis expressed in the vascular endothelium. The vascular endotheliumrefers to the endothelial cells lining blood vessels. It is understoodthat these blood vessels innervate the organs and tissues of the presentinvention. In a specific embodiment, the transgene is expressed invascular endothelium of a tissue or organ selected from the groupincluding but not limited to: heart, kidney, liver, lung, cornea andblood vessels. The expression can be at any level, but in a specificembodiment, the expression is at a high level. In one embodiment,expression of transgenes described herein is driven by anendothelial-specific promoter. In a specific embodiment, theendothelial-specific promoter is intercellular adhesion molecule 2(ICAM-2). In another specific embodiment, the endothelial-specificpromoter is TIE-2. In certain embodiments, the promoter is a porcinepromoter.

An anticoagulant according to the present invention can be selected fromthe group that includes tissue factor pathway inhibitor (TFPI), hirudin,thrombomodulin (TM), endothelial protein C receptor (EPCR), and CD39. Ina particular embodiment, the anticoagulant is TFPI. In anotherembodiment, the anticoagulant is CD39. In another embodiment, theanticoagulant is thrombomodulin.

An immunomodulator according to the present invention can be acomplement inhibitor or an immunosuppressant. In specific embodiments,the immunomodulator is a complement inhibitor. The complement inhibitorcan be CD46 (or MCP), CD55, CD59 and/or CR1. In a specific embodiment,at least two complement inhibitors can be expressed. In one embodiment,the complement inhibitors can be CD55 and CD59. In another embodiment,the immunomodulator can be a class II transactivator or mutants thereof.In certain embodiments, the immunomodulator can be a class IItransactivator dominant negative mutant (CIITA-DN). In another specificembodiment, the immunomodulator is an immunosuppressant. Theimmunosuppressor can be CTLA4-Ig. Other immunomodulators can be selectedfrom the group but not limited to CIITA-DN, PDL1, PDL2, or tumornecrosis factor-α-related apoptosis-inducing ligand (TRAIL), Fas ligand(FasL, CD95L) CD47, known as integrin-associated protein (CD47), HLA-E,HLA-DP, HLA-DQ, and/or HLA-DR.

The cytoprotective transgene according to the present invention can bean anti-apoptotic, an anti-oxidant or an anti-inflammatory transgene. Incertain embodiments, the cytoprotective transgene is selected from thegroup that includes A20, HO-1, FAT-1, catalase, and soluble TNF-alphareceptor (sTNFR1).

In a specific embodiment, the present invention provides porcineanimals, tissues and cells with at least the following geneticmodifications: lack of expression of GT, expression of CD46 andendothelial-specific expression of thrombomodulin. In a particularembodiment, CD46 is ubiquitously expressed.

In another embodiment, the present invention provides porcine animals,tissues and cells with at least the following genetic modifications:lack of expression of GT, expression of a complement inhibitor,endothelial-specific expression of an anticoagulant and/orendothelial-specific expression of an immunomodulator. In anotherspecific embodiment, the present invention provides porcine animals,tissues and cells with at least the following genetic modifications:lack of expression of GT, expression of CD46, and endothelial-specificexpression of thrombomodulin. In a further embodiment, the presentinvention provides porcine animals, tissues and cells with at least thefollowing genetic modifications: lack of expression of GT, expression ofCD46 and endothelial-specific expression of CD39. In a specificembodiment, the present invention provides porcine animals, tissues andcells with at least the following genetic modifications: lack ofexpression of GT, expression of CD46, endothelial-specific expression ofthrombomodulin, and endothelial-specific expression of CD39. Inparticular embodiments, CD46 can be ubiquitously expressed.

In another specific embodiment, the present invention provides porcineanimals, tissues and cells with at least the following geneticmodifications: lack of expression of GT, expression of CD46,endothelial-specific expression of thrombomodulin, andendothelial-specific expression of CTLA4-Ig. In a further specificembodiment, the present invention provides porcine animals, tissues andcells with at least the following genetic modifications: lack ofexpression of GT, expression of CD46, endothelial-specific expression ofthrombomodulin, and endothelial-specific expression of CIITA-DN. In aparticular embodiment, CD46 is ubiquitously expressed.

In another specific embodiment, the present invention provides porcineanimals, tissues and cells with at least the following geneticmodifications: lack of expression of GT, expression of CD46,endothelial-specific expression of thrombomodulin, and expression ofEPCR. In a particular embodiment, CD46 is ubiquitously expressed. In oneembodiment, expression of EPCR is driven by a endothelial-specificpromoter. In a specific embodiment, the endothelial-specific promoter isporcine ICAM-2. In another specific embodiment, the endothelial-specificpromoter is TIE-2. In one embodiment, expression of EPCR is driven by aubiquitous promoter. In a specific embodiment, the ubiquitous promoteris CAG.

In a further specific embodiment, the present invention provides porcineanimals, tissues and cells with at least the following geneticmodifications: lack of expression of GT, expression of CD46,endothelial-specific expression of thrombomodulin, endothelial-specificexpression of CD39, and endothelial-specific expression of CTLA4-Ig. Ina particular embodiment, CD46 is ubiquitously expressed. In an alternateembodiment, the porcine can express TFPI in place of or in addition tothrombomodulin.

In another specific embodiment, the present invention provides porcineanimals, tissues and cells with at least the following geneticmodifications: lack of expression of GT, expression of CD46, expressionof an cytoprotective transgene, endothelial-specific expression ofthrombomodulin, endothelial-specific expression of CD39, andendothelial-specific expression of CTLA4-Ig. In a particular embodiment,CD46 is ubiquitously expressed.

In another specific embodiment, the present invention provides porcineanimals, tissues and cells with at least the following geneticmodifications: lack of expression of GT, expression of CD46, expressionof a cytoprotective transgene, endothelial-specific expression ofthrombomodulin and endothelial-specific expression of CD39. In aparticular embodiment, CD46 is ubiquitously expressed. In an alternateembodiment, the porcine can express TFPI in place of or in addition tothrombomodulin.

In a further specific embodiment, the present invention provides porcineanimals, tissues and cells with at least the following geneticmodifications: lack of expression of GT, expression of CD46, expressionof CIITA-DN, and endothelial specific expression of thrombomodulinand/or endothelial specific expression of CD39. In a particularembodiment, CD46 is ubiquitously expressed.

In a further specific embodiment, the present invention provides porcineanimals, tissues and cells with at least the following geneticmodifications: lack of expression of GT, expression of CD46, expressionof DAF, expression of CIITA-DN, and endothelial-specific expression ofthrombomodulin and/or endothelial-specific expression of CD39.Alternately, the present invention provides porcine animals, tissues andcells with at least the following genetic modifications: lack ofexpression of GT, expression of CD46, endothelial-specific expression ofthrombomodulin and/or endothelial-specific expression of EPCR. In afurther specific embodiment, the present invention provides porcineanimals, tissues, particularly lungs, and cells, particularly lungcells, with at least the following genetic modifications: lack ofexpression of GT, expression of CD46, and endothelial-specificexpression of thrombomodulin. In a particular embodiment, CD46 isubiquitously expressed.

In one embodiment, a method is provided for treatment or prophylaxis oforgan dysfunction including administering the organs or cells of thepresent invention to a host in need thereof. In a particular embodiment,the host has heart, liver, kidney or lung dysfunction. In anotherembodiment, the host is administered a vascularized xenograft and/or isderived from an organ, tissue or cell therefrom.

In one embodiment, the host is a primate. In a particular embodiment,the host is a human. In a specific embodiment, the host is a humansuffering from organ dysfunction.

In one embodiment, the organ is a porcine heart. In another embodiment,the organ is a porcine kidney. In another embodiment, the organ is aporcine lung. In another embodiment, the organ in is a porcine liver. Inanother embodiment, the cells are porcine liver-derived cells, livertissue slices; or isolated liver cells. In a particular embodiment, thecells are porcine hepatocytes. In a particular embodiment porcinehepatocytes or porcine liver tissue slices can be used in a medicaldevice. In additional embodiments, organs according to the presentinvention can be selected from the following: heart, lung, liver,kidney, intestine, spleen, and pancreas. In one embodiment, the organscan be used as bridge organs until a human organ becomes available. Inother embodiments, the xenotransplanted organs of the present inventioncan survive and function in the recipient like an allograft.

In a particular embodiment, a porcine donor liver may be used as abridge transplant, allowing for stabilization of a patient until a humandonor liver (allograft) becomes available.

It is envisioned that in certain embodiments of the present invention,the endothelial cells themselves produced by the methods disclosedherein can be used as the xenotransplanted cell.

In a particular embodiment, porcine endothelial cells from the corneacan be used as a transplant material to treat cornea dysfunction.

In a particular embodiment, porcine endothelial cells from the retinacan be used as a transplant material to treat retina dysfunction.

In further embodiments, it is envisioned that the vasculature itself canbe used as the xenograft as a vascular graft. In some embodiments,vessels can be used as grafts for the following including but notlimited to vascular reconstructive surgery, coronary bypass surgery, orperipheral bypass surgery to treat atherosclerosis, coronary arterydisease, peripheral vascular disease or aortic aneurysm. In embodimentsof the present invention, vessels can include large and microvasculaturetissue, such as microvessels, capillaries, microcappilaries andcapillary beds.

In one embodiment, the dose of immunosuppressive drug(s)/agent(s) is/arereduced compared to other methods. In a specific embodiment, the dosageof one or more of daclizumab, tacrolimus, and/or sirolimus is reducedcompared to dosages used in other methods of transplantation. Inparticular embodiments of the present invention, clinically relevantimmunosuppressant regimens are provided in conjunction with the organs,tissues and cells described herein.

In another embodiment, the number of types of immunosuppressivedrug(s)/agent(s) is/are reduced compared to other methods.

In one embodiment, the duration of immunosuppression is shortenedcompared to other methods.

In another embodiment, lower or no maintenance immunosuppression is usedcompared to other methods.

In one embodiment, a method is provided for treatment or prophylaxis oforgan dysfunction including administering the organs, tissues or cellsof the present invention to a host, wherein post-transplant there arenot numerous, or serious life-threatening, complications associated withone or more of the transplant procedure, the immunosuppressive regime orthe tolerance inducing regime. In one embodiment, a method is providedfor treatment or prophylaxis of organ dysfunction includingadministering the organs, tissues or cells of the present invention to ahost, wherein post-transplant there are not numerous, or seriouslife-threatening, complications associated with one or more of thetransplant procedures. In a specific embodiment, a method is providedfor treatment or prophylaxis of organ dysfunction includingadministering the organs, tissues or cells of the present invention to ahost, wherein post-transplant there are not numerous, or seriouslife-threatening, complications, including consumptive coagulopathy.

In another embodiment, a method is provided for treatment or prophylaxiseye disease, including for treatment of cornea or retina dysfunction,including administering the organs, tissues or cells of the presentinvention to a host, wherein post-transplant there are not numerous, orserious life-threatening, complications associated with one or more ofthe transplant procedure, the immunosuppressive regime or the toleranceinducing regime.

Other embodiments of the present invention will be apparent to one ofordinary skill in light of the following description of the invention,the claims and what is known in the art.

DESCRIPTION OF FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1a and 1b are representative figures of the vectors used in theinvention.

FIG. 1a : vector pREV859B is the Tie-2 promoter/enhancer linked to aCD39 transgene; vector pREV861 is the ICAM-2 promoter/enhancer linked toa CD39 transgene.

FIG. 1b : vector pREV 871 is the ICAM-2 promoter/enhancer linked to aTFPI transgene; vector pREV 872 is the is the ICAM-2 promoter/enhancerlinked to a TM transgene; vector pREV 873 is the ICAM-2promoter/enhancer linked to an EPCR transgene.

FIG. 2 shows flow cytometric analysis of transgenic protein expressionin transfected porcine immortal aortic endothelial cells (AOCs).Anti-human thrombomodulin (TM) monoclonal antibody (mAb) reacted with(A), non-transfected AOCs and (B) AOC's transfected with human TM andhuman EPCR. Anti-human endothelial protein C receptor (EPCR) mAb reactedwith (C) non-transfected AOC's and (D) AOCs transfected with human TMand human EPCR.

FIG. 3 shows flow cytometric analysis of transgenic protein expressionin endothelial cells isolated from piglets 440-04 (CD39 transgenic) and424-01 (TM transgenic), stained with anti-CD39 and anti-CD141(TM),respectively. (A) shows isotype control binding to endothelial cellsfrom piglet 440-4. (B) shows anti-human CD39 monoclonal antibody (mAb)binding to CD39 positive endothelial cells from 440-4. (C) shows isotypecontrol binding to endothelial cells from piglet 424-01. (D) showsanti-human CD141 (TM) mAb binding to TM positive endothelial cells from424-01.

FIG. 4 presents images of cells stained with FITC labeled anti-human TMantibody. TM expression was observed in the endothelium of a vessel froma tail biopsy of piglet 424-03. The background fluorescence (BF) showsvessel morphology. Isotype control is also shown.

FIG. 5 shows TM transcript expression by RTPCR in samples obtained frommulti-transgenic piglets 448-01, 448-02, 448-03 and 450-06. TM copynumber shown is the copy number of hTM present in 50 ng of cDNA.

DETAILED DESCRIPTION OF THE INVENTION

The immunobiology of xenotransplantation, between discordant species,has been well detailed in the pig-to-primate model (reviewed by Ekserand Cooper, 2010 Expert Rev. Clin. Immuol. 6(2):219-230; Li et al.,Transpl Immunol. 2009 Jun.; 21(2):70-4; Le Bas-Bernardet and BlanchoTranspl Immunol. 2009 Jun.; 21(2):60-4; Pierson et al.,Xenotransplantation. 2009 September-October; 16(5):263-80). In initialstudies, wild-type pig organs transplanted into non-human primates wererejected due to the binding of natural (preformed) antibodies to the pigvascular endothelium and initiation of the complement cascade.Endothelial cells responded to this immune activation by converting froman anticoagulant to a coagulant phenotype, and HAR resulted (Robson etal., Int Arch Allergy Immunol. 1995 April; 106(4):305-22.). The removalof the Gal epitope from the cell surface in genetically engineered “Galknock-out” (GTKO) pigs eliminated HAR (Kuwaki et al., Nat Med. 2005Jan.; 11(1):29-31). Subsequently, studies utilizing GTKO pigs identifiedfurther forms of xenorejection, characterized by intravascularcoagulation, and thrombosis in the graft. The first, termed acutehumoral xenograft rejection (AHXR) or delayed xenograft rejection (DXR),is triggered by either antibody/cell-mediated damage of the endotheliumor by coagulation factor incompatibilities between the discordantspecies (pig and non-human primate), leading to endothelial activation.Once activated, the endothelium changes from its anticoagulant state toa procoagulant state by up regulation of von Willebrand factor andproduction of tissue factor leading to thrombus formation, hemorrhage,and rejection of the graft (Mohiuddin, 2007, PLoS Medicine, Vol 4(3) p.0429-0434). In addition to AHXR, in the absence of intenseimmunosuppression regimes, xenografts may undergo acute cellularrejection, characterized by T- and B-cell infiltration of the graft andT-cell activation (Ekser and Cooper, 2010). Therefore, in thischallenging endothelial environment, a xenograft must be capable ofpreventing or dampening all of these immunological responses, to remainviable and functional. Expression of multiple transgenes, such asanticoagulants, immunosuppressant and cytoprotective transgenes, in atissue specific manner within the porcine endothelium of a xenografts,on a GTKO genetic background, will address these multiple immunologicalchallenges. Therefore, the present invention provides geneticallyengineered pigs with the GTKO genetic background plus other transgenestowards improved outcomes in organ, tissue or endothelial cellxenotransplantation. Organs, tissues and cells from GTKO pigs expressingother transgenes specifically in endothelium, will provide significantprotection of the xenografted material from the recipient's immuneresponse.

A “transgene” is a gene or genetic material that has been transferredfrom one organism to another. Typically, the term describes a segment ofDNA containing a gene sequence that has been isolated from one organismand is introduced into a different organism. This non-native segment ofDNA may retain the ability to produce RNA or protein in the transgenicorganism, or it may alter the normal function of the transgenicorganism's genetic code. In general, the DNA is incorporated into theorganism's germ line. For example, in higher vertebrates this can beaccomplished by injecting the foreign DNA into the nucleus of afertilized ovum. When inserted into a cell, a transgene can be either acDNA (complementary DNA) segment, which is a copy of mRNA (messengerRNA), or the gene itself residing in its original region of genomic DNA.The transgene can be a genome sequence, in particular when introduced aslarge clones in BACs (bacterial artificial chromosomes) or cosmid.Transgene “expression” in the context of the present specification,unless otherwise specified, means that a peptide sequence from anon-native nucleic acid is expressed in at least one cell in a host. Thepeptide can be expressed from a transgene that is incorporated in thehost genome.

A “donor” is meant to include any non-human organism that may serve as asource of donor tissue or cells for xenotransplantation including, butnot limited to, mammals, birds, chickens, reptiles, fish, and insects.The donor may be in any stage of development, including, but not limitedto fetal, neonatal, young and adult. An “animal” is typically a mammal.A “mammal” is meant to include any non-human mammal, including but notlimited to pigs, sheep, goats, cattle (bovine), deer, mules, horses,monkeys, dogs, cats, rats, and mice. In one embodiment of the invention,genetically altered pigs and methods of production thereof are provided.The animals of the invention are “genetically modified” or “transgenic,”which means that they have a transgene, or other foreign DNA, added orincorporated, or an endogenous gene modified, including, targeted,recombined, interrupted, deleted, disrupted, replaced, suppressed,enhanced, or otherwise altered, to mediate a genotypic or phenotypiceffect in at least one cell of the animal, and typically into at leastone germ line cell of the animal. In some embodiments, animals may havethe transgene integrated on one allele of its genome (heterozygoustransgenic). In other embodiments, animals may have the transgene on twoalleles (homozygous transgenic).

The term “ungulate” refers to hoofed mammals. Artiodactyls are even-toed(cloven-hooved) ungulates, including antelopes, camels, cows, deer,goats, pigs, and sheep. Perissodactyls are odd toes ungulates, whichinclude horses, zebras, rhinoceroses, and tapirs. The term ungulate asused herein refers to an adult, embryonic or fetal ungulate animal.

The terms “porcine”, “porcine animal”, “pig” and “swine” are genericterms referring to the same type of animal without regard to gender,size, or breed.

The “cells” “tissues” and “organs” of the invention are derived from ananimal. Although the cells, tissues and organs can be derived from amature animal, in some embodiments the cells, tissues and organs arederived from a fetal or neonatal tissue. In particular embodiments ofthe invention, the cells, tissues and organs, are derived from atransgenic porcine animal and in particular, a transgenic porcine thathas grown to a sufficient size to be useful as a transplant donor. Incertain embodiments, the animals survive past weaning age. In specificembodiments, the animals are at least six months old. In certainembodiments, the animal survives to reach breeding age. In certainembodiments, the animal is a porcine animal of at least 300 pounds. Inspecific embodiments, the animal is a porcine sow and has given birth atleast one time.

“High” levels of expression are considered sufficient to provide aphenotype (detectable expression or therapeutic benefit). Typically a‘high’ level of expression is sufficient to be capable of imparting aphenotypic or therapeutic benefit to the animal. For example, it can becapable of reducing graft rejection including hyperacute rejection(HAR), acute humoral/vascular xenograft rejection (AHXR/AVXR), and Tcell-mediated cellular rejection. It was previously unknown whetheranticoagulant and immunosuppressive transgenes could be expressed inporcine endothelium at levels capable of reducing these types ofrejection.

The “endothelium” is an epithelium of mesoblastic origin composed of asingle layer of thin flattened cells that lines internal body cavities.For example, the serous cavities or the interior of the heart contain anendothelial cells lining and the “vascular endothelium” is theendothelium that lines blood vessels. (Medline Plus, National Library ofMedicine)

The term “clinically relevant immunosuppressive regimen” refers to aclinically acceptable regimen of immunosuppressant drugs provided to apatient following organ, tissue or cell transplantation of a geneticallymodified pig as disclosed herein. Determining clinical relevancerequires a judgment call generally by the FDA balancing acceptable riskversus potential benefit such that human safety is preserved while theefficacy of the drug or treatment is maintained. In one example, the FDAcan examine the number of adverse event associated with a particularregimen. An adverse event is any unfavorable and unintended sign(including an abnormal laboratory finding, for example), symptom, ordisease temporally associated with the use of a medicinal product,whether or not considered related to the medical product.

As used herein, the terms “endothelial-specific”, “specific transgeneexpression in endothelial tissue”, “specifically expresses at least onetransgene in endothelial tissue” and the like, it is understood thatthese terms refer to a transgene under control of a endothelial-specificregulatory element that allows for the restricted expression of atransgene in endothelial tissue and/or cells. The transgene function andexpression is restricted to endothelial tissue and/or cells.

“Endothelial-specific regulatory element” and the like refer to apromoter, enhancer or a combination thereof wherein the promoter,enhancer or a combination thereof drives restricted expression of atransgene in endothelial tissue and/or cells. The regulatory elementprovides transgene function and expression restricted to endothelialtissue and/or cells.

Transgenic Animals

In one embodiment, porcine animals, organs, tissues and cells areprovided that have at least four genetic modifications. Such geneticmodifications can include, without limitation, additions and/ordeletions of genes, including knock-outs and knock-ins, knock-down, aswell as re-arrangements. In a particular embodiment, porcine animals,organs, tissues and cells are provided that have at least three or atleast four genetic modifications, wherein at least one, at least two, atleast three or four of the genetic modifications are transgenes and atleast one, at least two, at least three or four of the transgenes areubiquitously expressed. In a particular embodiment, porcine animals,organs, tissues and cells are provided that have at least four geneticmodifications, wherein at least one genetic modification is a knock-out.

In a particular embodiment, porcine animals, tissues organs, and cellsare provided that have at least one gene knocked out and express atleast three transgenes. In a specific embodiment, the at least one geneis knocked out by homologous recombination.

In one embodiment, porcine animals, organs, tissues and cells areprovided that have at least five genetic modifications. Such geneticmodifications can include, for example, additions and/or deletions ofother genes, including knock-outs and knock-ins, as well asrearrangements. In a particular embodiment, porcine animals, organs,tissues and cells are provided that have at least five geneticmodifications, wherein at least one, at least two, at least three, atleast four or five of the genetic modifications are transgenes and atleast one, at least two, at least three, at least four or five of thetransgenes are ubiquitously expressed. In a particular embodiment,porcine animals, organs, tissues and cells are provided that have atleast five genetic modifications, wherein at least one geneticmodification is a knock-out.

In a particular embodiment, porcine animals, tissues and cells areprovided that have at least one gene knocked out and express at leastfour transgenes. In a specific embodiment, the at least one gene isknocked out by homologous recombination.

In one embodiment, porcine animals, organs, tissues and cells areprovided that lack any expression of functional alpha 1,3galactosyltransferase (GTKO) and express at least one transgene inendothelium. In other embodiments, GTKO animals, organs, tissues andcells are provided which express multiple transgenes in endothelium. Inparticular subembodiments, the animals, tissues and cells express atleast one immunomodulator. In certain embodiments, the animals, organs,tissues and cells express more than one immunomodulator. In particularembodiments, GTKO animals, organs, tissues and cells are provided thatexpress at least one immunomodulator and at least one anticoagulanttransgene. In one embodiment, the immunomodulator is animmunosuppressant. In an alternate embodiment, the immunomodulator is acomplement inhibitor. In a particular embodiment, expression of theimmunomodulator is specific to the endothelium. In a further particularembodiment, expression of the immunosuppressant is specific to theendothelium. In a still further specific embodiment, expression of thecompliment inhibitor is specific to the endothelium. In othersubembodiments, the animals, organs, tissues and cells express at leastone anticoagulant. In certain embodiments, the animals, organs, tissuesand cells express more than one anticoagulant. In a particularembodiment, the expression of the anticoagulant is specific to theendothelium. In one subembodiment, the animals, organs, tissues andcells express at least one cytoprotective transgene. In anotherembodiment, the animals, organs, tissues and cells express more than onecytoprotective transgene. In one embodiment, the transgene isspecifically expressed in endothelium.

In one embodiment, the present invention includes GTKO animals, organs,tissues and cells that lack any expression of functional alpha 1,3galactosyltransferase (GTKO) and expresses at least one complimentinhibitor and at least one additional transgene selected from the groupconsisting of anticoagulants, immunosuppressants and cytoprotectants. Ina particular embodiment, the expression of the at least one additionaltransgene is specific to the endothelium.

In a specific embodiment, GTKO animals, organs, tissues and cells areprovided that express at least one compliment inhibitor (e.g., CD46) andat least one anticoagulant (e.g., thrombomodulin).

In another specific embodiment, GTKO animals, organs, tissues and cellsare provided that express at least one compliment inhibitor (e.g., CD46)and at least two anticoagulants (e.g., thrombomodulin and CD39).

In another specific embodiment, GTKO animals, organs, tissues and cellsare provided that express at least one compliment inhibitor (e.g., CD46)and at least one immunosuppressant (e.g., CTLA4).

In a still further specific embodiment, GTKO animals, organs, tissuesand cells are provided that express at least one compliment inhibitor(e.g., CD46) and a cytoprotective transgene (e.g., A20).

In certain embodiments, GTKO animals, organs, tissues and cells areprovided that express at least one immunosuppressant, at least onecomplement inhibitor and at least one anticoagulant transgene. In anfurther particular embodiment, GTKO animals, organs, tissues and cellsare provided that express at least one immunosuppressant, at least onecomplement inhibitor and at least two anticoagulant transgenes. In aspecific embodiment, GTKO animals, organs, tissues and cells areprovided that express at least one immunosuppressant, at least onecomplement inhibitor and at least one anticoagulant transgenes, whereinexpression of the at least one immunosuppressant and the at least oneanticoagulant transgenes is specific to the endothelium. In yet anotherspecific embodiment, GTKO animals, organs, tissues and cells areprovided that express at least one immunosuppressant, at least onecomplement inhibitor and at least two anticoagulant transgenes, whereinexpression of the at least one immunosuppressant and the at least twoanticoagulant transgenes is specific to the endothelium. In oneembodiment, GTKO animals, organs, tissues and cells are provided thatexpress at least one immunomodulator, at least one anticoagulant and atleast one cytoprotective transgene. In a further embodiment, GTKOanimals, organs, tissues and cells are provided that express at leastone immunosuppressant, at least one complement inhibitor, at least oneanticoagulant transgene and at least one cytoprotective transgene. In afurther particular embodiment, GTKO animals, organs, tissues and cellsare provided that express at least one immunosuppressant, at least onecomplement inhibitor, at least two anticoagulant transgenes and at leastone anti-cytoprotective transgene. In a particular embodiment, GTKOanimals, organs, tissues and cells are provided that express at leastone immunosuppressant, at least one complement inhibitor, at least oneanticoagulant transgene and at least one cytoprotective transgene,wherein the expression of the at least one immunosuppressant and the atleast one anticoagulant transgenes is specific to the endothelium. In aparticular embodiment, GTKO animals, organs, tissues and cells areprovided that express at least one immunosuppressant, at least onecomplement inhibitor, at least two anticoagulant transgenes and at leastone cytoprotective transgene, wherein the expression of the at least oneimmunosuppressant and the at least two anticoagulant transgenes isspecific to the endothelium. In a specific embodiment, the expression ofthe anti-apoptotic transgene is specific to the endothelium.

In one embodiment, the transgenic porcine animals described herein areviable. In another embodiment, the animals described herein are fertile.In further embodiments, the animals described herein can stably transmitsome of its genetic modifications to its offspring. In still furtherembodiments, the animals described herein can stably transmit all of itsgenetic modifications to its offspring. In certain embodiments, theanimals can stably transmit all of its genetic modifications to itsoffspring when the animals are bred naturally. In other embodiments, themultiple transgenes exhibit co-segregation to offspring. In a particularembodiment, porcine animal, organs, tissues and cells are provided withat least the following genetic modifications: lack of expression of GT,expression of a complement inhibitor, endothelial-specific expression ofan anticoagulant transgene, and endothelial-specific expression of animmunosuppressant transgene. In a particular embodiment, porcineanimals, organs, tissues and cells are provided with at least thefollowing genetic modifications: lack of expression of GT, expression ofa complement inhibitor, endothelial-specific expression of twoanticoagulant transgenes, and expression of an immunosuppressanttransgene. In another embodiment, porcine animals, organs, tissues andcells are provided with at least the following genetic modifications:lack of expression of GT, expression of a complement inhibitor,expression of a cytoprotective transgene, endothelial-specificexpression of an anticoagulant transgene, and expression of animmunosuppressant transgene. In a particular embodiment, porcineanimals, organs, tissues and cells are provided with at least thefollowing genetic modifications: lack of expression of GT, expression ofa complement inhibitor, expression of a cytoprotective transgene,endothelial-specific expression of two anticoagulant transgenes, andexpression of an immunosuppressant transgene. In a specific embodiment,the expression of the cytoprotective transgene is alsoendothelium-specific. An immunomodulator can be a complement inhibitoror an immunosuppressant. In specific embodiments, the immunomodulator isa complement inhibitor. The complement inhibitor can be CD46 (or MCP).In other embodiments, the complement inhibitor is CD55, CD59 or CR1. Incertain embodiments, the transgene is expressed from a ubiquitouspromoter. In certain other embodiments, the transgene is expressed froma promoter active primarily in endothelium. The expression can be at anylevel, but in specific embodiments, the expression is at high levels.Typically a ‘high’ level of expression is sufficient to be capable ofimparting a phenotypic or therapeutic benefit to the animal.

An immunomodulator can also be an immunosuppressant. Theimmunosuppressant can be capable of down-regulating a T-cell mediatedresponse. In particular, the immunosuppressant can be CTLA4-Ig ormutants thereof. In other embodiments, the immunosuppressant transgeneis a ligand that interferes with CD28 activity, such as a B7 receptorpeptide or mutant thereof. In certain embodiments, the transgene isexpressed from a promoter active primarily in endothelium. Theexpression can be at any level, but in specific embodiments, theexpression is at high levels.

In other embodiments, the immunomodulator can be selected from the groupthat includes class II transactivators (CIITA) and mutants, includingdominant negative mutants thereof (CIITA-DN), PDL1, PDL2, tumor necrosisfactor-α-related apoptosis-inducing ligand (TRAIL), Fas ligand (FasL,CD95L) integrin-associated protein (CD47), HLA-E, HLA-DP, HLA-DQ, orHLA-DR. In certain other embodiments, the transgene is expressed from apromoter active primarily in endothelium. In certain embodiments, theimmunomodulator transgene is expressed from a ubiquitous promoter. Theexpression can be at any level, but in specific embodiments, theexpression is at high levels.

In one embodiments, the anticoagulant is selected from the group thatincludes tissue factor pathway inhibitor (TFPI), hirudin,thrombomodulin, endothelial protein C receptor (EPCR), and CD39. In aparticular embodiment, the anticoagulant is thrombomodulin. In anotherparticular embodiment, the anticoagulant is CD39. In certain otherembodiments, the transgene is expressed from a promoter active primarilyin endothelium. The expression can be at any level, but in specificembodiments, the expression is at high levels.

The cytoprotective transgene can be an anti-apoptotic, anti-oxidant oranti-inflammatory transgene. In certain embodiments, the cytoprotectivetransgene is selected from the group that includes A20, HO-1, FAT-1,catalase, and soluble TNF-alpha receptor (sTNFR1). In certain otherembodiments, the transgene is expressed from a promoter active primarilyin endothelial cells. The expression can be at any level, but inspecific embodiments, the expression is at high levels.

In certain embodiments, the one or more immunosuppressant oranticoagulant transgenes is expressed in the endothelium of tissues ofGTKO porcine animals which express high levels of CD46. In particularembodiments, porcine animals, tissues and cells are provided derivedfrom GTKO animals that express high levels of CD46 and expressthrombomodulin in endothelium. In a separate embodiment, porcineanimals, tissues and cells derived from GTKO animals are provided thatexpress high levels of CD46 and express CD39 in endothelium. In afurther embodiment, porcine animals, tissues and cells derived from GTKOanimals are provided that express high levels of CD46 and express CD39and/or thrombomodulin in endothelium.

In some embodiments, the immunomodulator has the sequence of a humanprotein. In other embodiments, the immunomodulator has the sequence of aporcine protein. In some embodiments, the anticoagulant has the sequenceof a human protein. In other embodiments, the anticoagulant has thesequence of a porcine protein. In some embodiments, the cytoprotectivetransgene has the sequence of a porcine protein. In another embodiment,the cytoprotective transgene has the sequence of a human protein. Inparticular embodiments, the porcine animal, organ, tissue or cellexpresses a human CD46 transgene. In particular embodiments, the porcineanimal, organ, tissue or cell expresses a human CTLA4-Ig transgene. Incertain embodiments, the porcine animal, organ, tissue or cell expressesa human thrombomodulin. In certain embodiments, the porcine animal,organ, tissue or cell expresses a human CD39. In certain embodiments,the porcine animal, organ, tissue or cell expresses a human TFPI. Inparticular embodiments, the porcine animal, tissue or cell expresses aporcine CTLA4 transgene. In a particular embodiment, porcine animals,organs, tissues and cells are provided with at least the followinggenetic modifications: lack of expression of GT, expression of CD46,endothelial-specific expression of TFPI, and endothelial-specificexpression of CTLA4-Ig. In another particular embodiment, porcineanimals, organs, tissues and cells are provided with at least thefollowing genetic modifications: lack of expression of GT, expression ofCD46, endothelial-specific expression of TFPI, endothelial-specificexpression of CD39, and endothelial-specific expression of CTLA4-Ig. Ina particular embodiment, the CD46 can be a human CD46. In anotherparticular embodiment, the human CD46 can be expressed at high levels.

In another particular embodiment, porcine animals, organs, tissues andcells are provided with at least the following genetic modifications:lack of expression of GT, expression of CD46, expression of acytoprotective transgene, endothelial-specific expression ofthrombomodulin, and endothelial-specific expression of CTLA4-Ig. Inanother particular embodiment, porcine animal, tissues and cells areprovided with at least the following genetic modifications: lack ofexpression of GT, expression of CD46, expression of a cytoprotectivetransgene, endothelial-specific expression of thrombomodulin,endothelial-specific expression of CD39, and endothelial-specificexpression of CTLA4-Ig.

In another particular embodiment, porcine animals, organs, tissues andcells are provided with at least the following genetic modifications:lack of expression of GT, expression of CD46, endothelial-specificexpression of thrombomodulin and/or CD39, and expression of CIITA.

In another particular embodiment, porcine animal, tissues and cells areprovided with at least the following genetic modifications: lack ofexpression of GT, expression of CD46, expression of DAF,endothelial-specific expression of thrombomodulin and/or CD39, andexpression of CIITA.

In certain embodiments, the transgene is expressed from a promoteractive primarily in endothelial cells (EC) (“endothelium specificpromoters”). Endothelium specific promoters of the present inventioninclude, but are not limited to: vascular cell adhesion molecule-1(VCAM-1), von Willebrand factor (vWF), endothelial nitric oxide synthase(eNOS), tyrosine kinase (Tie), fms-like tyrosine kinase-1 (FLT-1),kinase domain receptor (KDR/flk-1), intercellular adhesion molecule-2(ICAM-2) and endoglin. (For example, all reviewed (for use in adenoviralgene transfer vectors) by Beck et al., Current Gene Therapy, 2004, 4,457-467, Table 2A.) Others promoters which can be used for expression oftransgenes in the vasculature include but are not limited to CD31(platelet endothelial cell adhesion molecule [PECAM]) promoter,Pre-Proendothelin-1 (PPE-1) Promoter (see, for example, U.S. Pat. No.5,747,340 and U.S. Patent Publication No. 2007/0286845), and LDL LOX-1(White et al., Gene Ther. 2008 Mar.; 15(5):340-6; which targets thearterial vasculature). CD31 (platelet endothelial cell adhesion molecule[PECAM]) promoter limits expression to endothelial cells, monocytes, andplatelets and has been used to target hirudin and TFPI to activatedendothelium in transgenic mice (Chen et al., Blood. 2004 Sep. 1;104(5):1344-9). Also embodied herein are smooth muscle cell (SMC)promoters, which localize transgene expression in the smooth musclelayer of blood vessels, in close proximity to the vascular endothelium(for a list of SMC promoters, see for example Beck et al., see Table2B).

In certain embodiments the promoter is an endothelium specific promoterincluding but not limited to the Tie-2 promoter, the ICAM-2 promoter orthe PECAM promoter. The promoters of the present invention can be from avertebrate animal, including but not limited to fish or mammalianpromoters such as tilapia, human, pig, rat, or mouse. In specificembodiments, the promoter is an ICAM-2 promoter from a vertebrateanimal, including but not limited to fish or mammalian promoters such astilapia, human, pig, rat, or mouse. In specific embodiments, thepromoter is the mouse Tie-2 promoter. In specific embodiments, thepromoter is the porcine ICAM-2 promoter.

In certain embodiments additional regulatory elements can beincorporated into the transgene expression system, including enhancerelements. In one embodiment, the enhancer can be an endothelial-specificenhancer. The enhancers can be selected from but not limited to one ofthe following: Tie-2 enhancer; the ICAM-2 enhancer; the PECAM enhancer,the pdx-1 enhancer and the chicken actin enhancer. The enhancer can be,for example, a pdx-1 enhancer or a chicken actin enhancer, or can be aninsulator element for example, a chicken beta-globin insulator, forenhanced expression of the transgene (Chung J H, Bell A C, FelsenfeldG., Proc Natl Acad Sci USA. 1997 Jan. 21; 94(2):575-80). In specificembodiments, the enhancer element used is the Tie-2 enhancer. Inspecific embodiments, the promoter is used in combination with anenhancer element that is a non-coding or intronic region of DNAintrinsically associated or co-localized with the promoter. Particularspecific embodiments of the present invention include the: Tie-2promoter combined with the Tie-2 enhancer; the ICAM-2 promoter combinedwith the ICAM-2 enhancer; the PECAM promoter with the PECAm enhancer;and/or any promoter disclosed herein combined with its intrinsicallyassociated enhancer element.

As used herein, the terms “endothelial-specific”, “specific transgeneexpression in endothelial tissue”, “specifically expresses at least onetransgene in endothelial tissue” and the like, it is understood thatthese terms refer to a transgene under control of anendothelial-specific regulatory element that allows for the restrictedexpression of a transgene in endothelial tissue and/or cells. Thetransgene function and expression is restricted to endothelial tissueand/or cells.

“Endothelial-specific regulatory element” and the like refer to apromoter, enhancer or a combination thereof wherein the promoter,enhancer or a combination thereof drives restricted expression of atransgene in endothelial tissue and/or cells. The regulatory elementprovides transgene function and expression restricted to endothelialtissue and/or cells.

In certain embodiments, the expression is restricted to endothelium andis not present in other porcine tissues. To analyze tissue specificexpression, one skilled in the art can use techniques to ascertain therelative expression pattern in endothelial tissues and cells versusother tissues and cells. In one embodiment, immunohistochemistry can beused to analyze endothelial-specific expression. In another embodiment,there will be immunohistochemical staining of cells containing thetransgene under control of endothelial-specific regulatory elementswhereas the cells without the transgene will not exhibit the staining.In another embodiment, real-time PCR can be used to analyzeendothelial-specific expression. In one embodiment, the number of copiesof amplified DNA from total RNA from cells containing the transgeneunder control of endothelial-specific regulatory elements will be atleast one logarithm higher than cells without the transgene. In anotherembodiment, flow cytometry can be used to analyze endothelial-specificexpression. In one embodiment, fluorescence intensity from cellscontaining the transgene under control of endothelial-specificregulatory elements will be approximately 95-100% whereas fluorescenceintensity form cells without the transgene will be approximately 0-5%.

In addition, expression can be present in fetal, neonatal, and maturetissues, each of which can be a source of donor material. In particularembodiments of the invention, the cells, and especially the endothelialcells, are derived from a transgenic porcine animal and in particular, atransgenic porcine that has grown to a sufficient size to be useful as adonor. In certain embodiments, the animals survive past weaning age. Inspecific embodiments, the animals are at least six months old. Incertain embodiments, the animal survives to reach breeding age. Incertain embodiments, the animal is a porcine animal of at least 300pounds.

In one embodiment, a method is provided for treatment or prophylaxis oforgan dysfunction including administering donor porcine tissues, organsor cells to a host suffering from organ dysfunction, wherein the porcinedonor material exhibits expresses at least one anticoagulant transgene.

In another embodiment, a method is provided for treatment or prophylaxisof cornea or retina dysfunction including administering donor porcinecorneal endothelial cells to a host suffering from eye disease,including cornea or retina dysfunction, wherein the porcine donormaterial exhibits expresses at least one anticoagulant transgene.

In one embodiment, the donor organ is a porcine heart. In anotherembodiment, the donor organ is a porcine kidney. In another embodiment,the donor organ is a porcine lung. In another embodiment, the donororgan in is a porcine liver. In another embodiment, the donor cells areporcine liver-derived cells, liver tissue slices; or isolated livercells. In a particular embodiment, the donor cells are porcinehepatocytes. In a particular embodiment porcine hepatocytes or porcineliver tissue slices may be used in a medical device.

In a particular embodiment, the porcine donor cells are endothelialcells from the cornea or retina used as a graft to treat cornea orretina dysfunction.

In another particular embodiment, the donor tissues are porcine bloodvessels or vascular tissues used as a graft, to treat vascular diseasesor defects.

In a further particular embodiment, the porcine donor cells areendothelial cells used to seed vascular grafts, or may be used forseeding during coronary procedures, such as stenting or bypass surgery.Vascular graft materials may be allografts (human origin), orbioengineered devices, or any other material used as a vascular graft.

In other embodiments, cells provided herein can be used in re-transplantprocedures.

In certain embodiments of the present invention, methods of treating orpreventing organ dysfunction in primates are provided involvingadministration of the organs, tissues or cells of the present inventionto primates in need thereof. In one embodiment, the primate is anon-human primate, in one non-limiting example, a monkey. In anotherembodiment, the primate is a human. In additional embodiments, theanimals can also contain genetic modifications to express animmunomodulator. The immunomodulator can be a complement pathwayinhibitor gene and in particular embodiments is selected from CD55,CD59, CR1 and CD46 (MCP). The complement inhibitor can be human CD46(hCD46) wherein expression is through a mini-gene construct (SeeLoveland et al., Xenotransplantation, 11(2):171-183. 2004). Theimmunomodulator can also be an immunosuppressor gene that has a T-cellmodulating effect—such as CTLA4-Ig, or a dominant negative inhibitor(downregulator) of class II MHC (CIITA), or other genes that modulatethe expression of B-cell or T cell mediated immune function. Transgenicpigs expressing a CIITA dominant negative mutant driven by a CAGpromoter have recently been produced and shown to have a down regulatedSLA class II expression (after cytokine stimulation) and a reduced humanT-cell response (see Hara et al., 2010 Am J Transplant. 2010 (Supplement4); 10:187. (Abstract 503). In further embodiments, such animals can befurther modified to eliminate the expression of genes which affectimmune function.

In additional embodiments, the animals can also contain geneticmodifications to express an anticoagulant. The anticoagulant mayinclude, but is not limited to, TFPI, hirudin, thrombomodulin, EPCR andCD39. In addition, the animals can be genetically modified to inhibitthe expression of a CMP-Neu5Ac hydroxylase gene (see, for example, U.S.Patent Publication. 2005-0223418), the iGb3 synthase gene (see, forexample, U.S. Patent Publication 2005-0155095), and/or the Forssmansynthase gene (see, for example, U.S. Patent Publication 2006-0068479).In addition, the animals can be genetically modified to reduceexpression of a pro-coagulant. In particular, in one embodiment, theanimals are genetically modified to reduce or eliminate expression of aprocoagulant gene such as the FGL2 (fibrinogen-like protein 2) (see, forexample, Marsden, et al. (2003) J din Invest. 112:58-66; Ghanekar, etal. (2004) J Immunol. 172:5693-701; Mendicino, et al. (2005)Circulation. 112:248-56; Mu, et al. (2007) Physiol Genomics.31(1):53-62).

In embodiments wherein a transgene is expressed, this expression may bevia a ubiquitous or tissue-specific promoter and may include additionalregulatory elements such as enhancers, insulators, matrix attachmentregions (MAR) and the like.

To achieve these additional genetic modifications, in one embodiment,cells isolated from a genetically modified pig can be further modifiedto contain multiple genetic modifications. In some embodiments thesecells can be used as donors to produce pigs with multiple geneticmodifications via nuclear transfer. In other embodiments, geneticallymodified animals can be bred together to achieve multiple geneticmodifications.

Transgenes to Target Acute Humoral Rejection

Xenografting is currently hindered by the severe and well-documentedproblems of rejection. This process can be divided into distinct stages,the first of which occurs within minutes of transplantation and iscalled “hyperacute rejection” (HAR). HAR is defined by the ubiquitouspresence of high titers of pre-formed natural antibodies binding to theforeign tissue. The binding of these natural antibodies to targetepitopes on the donor tissue endothelium is believed to be theinitiating event in HAR. This binding, within minutes of perfusion ofthe donor tissue with the recipient blood, is followed by complementactivation, platelet and fibrin deposition, and ultimately byinterstitial edema and hemorrhage in the donor organ, all of which causerejection of the tissue in the recipient (Strahan et al. (1996)Frontiers in Bioscience 1, e34-41). The primary course of HAR in humansis the natural anti-Gal antibody, which comprises approximately 1% ofantibodies in humans and monkeys.

This initial hyperacute rejection is then reinforced by the delayedvascular response (also known as acute humoral xenograft rejection(AHXR), acute vascular xenorejection (AVXR) or delayed xenograftrejection (DXR)). The lysis and death of endothelial cells during thehyperacute response is accompanied by edema and the exposure ofadventitial cells, which constitutively express tissue factor (TF) ontheir surface. Tissue factor is thought to be pivotal in the initiationof the in vivo coagulation cascade, and its exposure to plasma triggersthe clotting reactions. Thrombin and TNF-alpha become localized aroundthe damaged tissue and this induces further synthesis and expression ofTF by endothelial cells.

The environment around resting endothelial cells does not favorcoagulation. Several natural coagulation inhibitors are associated withthe extracellular proteoglycans of endothelial cells, such as tissuefactor pathway inhibitor, antithrombin III, and thrombomodulin. Therecognition of the foreign tissue by xenoreactive natural antibodies(XNAs), however, causes the loss of these molecules.

Together with the exposure and induction of tissue factor, theanticoagulant environment around endothelial cells thus becomespro-coagulant. The vascularised regions of the xenograft thus becomesites of blood clots, a characteristic of damaged tissue. Blood flow isimpaired and the transplanted organ becomes ischemic. A fuller accountof delayed vascular rejection can be found in Bach et al. (1996) ImmunolToday. 1996 Aug.; 17(8):379-84.

The present invention provides for animals, tissues or cells that may beused in xenotransplantation to produce low to no levels of one or moreof the following: HAR, AHXR/AVXR/DXR and/or ACXR. In one embodiment, theanimals, tissues or cells may be used in xenotransplantation to producelow to no levels of HAR and AHXR/AVXR. In another embodiment, theanimals, tissues or cells may be used in xenotransplantation to producelow to no levels of HAR, AHXR/AVXR and ACXR. As will be discussed indetail in the following sections, embodiments of the present inventioninclude various combinations of complement regulator expression,immunosuppressor expression, anticoagulant expression, and/or partiallyor fully depleted functional αGT expression in donor tissue.

In one embodiment, porcine animals, we well as organs, tissues and cellsthereof, are provided herein and express one or more transgenes. Inanother embodiment, porcine animals, we well as organs, tissues andcells thereof, are provided herein and express one or more transgenesselected from but not limited to the following: at least two transgenes,at least three transgenes, at least four transgenes, at least fivetransgenes, at least six transgenes, at least seven transgenes and atleast eight transgenes. In further embodiments, cells from the porcineanimals provided herein can elicit a decreased immune response by humanlymphocytes (MLR assay) to said porcine cells. In another embodiment,cells expressing transgenes are shown to inhibit clotting and thrombosiswhich occurs in the xenograft environment.

Alpha 1,3 Galactosyltransferase (αGT)

As noted previously, the primary course of HAR in humans is the naturalanti-galactose alpha 1,3-galactose (Gal) antibody, which comprisesapproximately 1% of IgG antibodies in humans and monkeys. Except for OldWorld monkeys, apes and humans, most mammals carry glycoproteins ontheir cell surfaces that contain the Gal epitope (Galili et al., J.Biol. Chem. 263: 17755-17762, 1988). Humans, apes and old world monkeysdo not express Gal, but rather produce in high quantities a naturallyoccurring anti-Gal antibody that causes an immediate hyperacute reactionupon xenotransplantation into humans of tissues from animals carryingthe Gal epitope (Sandrin et al., Proc Natl Acad Sci USA. 1993 Dec. 1;90(23):11391-5, 1993; review by Sandrin and McKenzie, Immunol Rev. 1994October; 141:169-90).

A variety of strategies have been implemented to eliminate or modulatethe anti-Gal humoral response caused by xenotransplantation, includingenzymatic removal of the epitope with alpha-galactosidases (Stone etal., Transplantation 63: 640-645, 1997), specific anti-gal antibodyremoval (Ye et al., Transplantation 58: 330-337, 1994), capping of theepitope with other carbohydrate moieties, which failed to eliminate αGTexpression (Tanemura et al., J. Biol. Chem. 27321: 16421-16425, 1998 andKoike et al., Xenotransplantation 4: 147-153, 1997) and the introductionof complement inhibitory proteins (Dalmasso et al., Clin. Exp. Immunol.86:31-35, 1991, Dalmasso et al. Transplantation 52:530-533 (1991)). C.Costa et al. (FASEB J 13, 1762 (1999)) reported that competitiveinhibition of αGT in transgenic pigs results in only partial reductionin epitope numbers. Similarly, S. Miyagawa et al. (J. Biol. Chem 276,39310 (2000)) reported that attempts to block expression of gal epitopesin N-acetylglucosaminyltransferase III transgenic pigs also resulted inonly partial reduction of gal epitopes numbers and failed tosignificantly extend graft survival in primate recipients.

Single allele knockouts of the αGT locus in porcine cells and liveanimals have been reported. Denning et al. (Nature Biotechnology 19:559-562, 2001) reported the targeted gene deletion of one allele of theαGT gene in sheep. Harrison et al. (Transgenics Research 11: 143-150,2002) reported the production of heterozygous αGT knock out somaticporcine fetal fibroblasts cells. In 2002, Lai et al. (Science 295:1089-1092, 2002) and Dai et al. (Nature Biotechnology 20: 251-255, 2002)reported the production of pigs, in which one allele of the αGT gene wassuccessfully rendered inactive. Ramsoondar et al. (Biol of Reproduc 69,437-445 (2003)) reported the generation of heterozygous αGT knockoutpigs that also express human alpha-1,2-fucosyltransferase (HT), whichexpressed both the HT and αGT epitopes. PCT publication No. WO 03/055302to The Curators of the University of Missouri confirms the production ofheterozygous αGT knockout miniature swine for use in xenotransplantationin which expression of functional αGT in the knockout swine is decreasedas compared to the wildtype.

PCT publication No. WO 94/21799 and U.S. Pat. No. 5,821,117 to theAustin Research Institute; PCT publication No. WO 95/20661 to Bresatec;and PCT publication No. WO 95/28412, U.S. Pat. Nos. 6,153,428, 6,413,769and US publication No. 2003/0014770 to BioTransplant, Inc. and TheGeneral Hospital Corporation provide a discussion of the production ofαGT negative porcine cells based on the cDNA of the αGT gene.

A recent, major breakthrough in the field of xenotransplantation was theproduction of the first live pigs lacking any functional expression ofαGT (Phelps et al. Science 299:411-414 (2003); see also PCT publicationNo. WO 04/028243 by Revivicor, Inc. and PCT Publication No. WO 04/016742by Immerge Biotherapeutics, Inc.).

In one embodiment, animals, tissues and cells are provided that lack anyexpression of functional αGT (GTKO) and express at least one additionaltransgene in endothelium. The additional transgene is typically selectedfrom: 1) an immunomodulator including a complement inhibitor (i.e. CD46(MCP), CD55, CD59, CR1 and the like) or an immunosuppressor (i.e.CTLA-4, B7 and the like) or 2) an anticoagulant (i.e. TFPI, hirudin,thrombomodulin, EPCR, CD39 and the like). In other embodiments, animals,tissue and cells are provided that lack any expression of functional αGTand express both at least one immunomodulator and at least oneanticoagulant in endothelium. Animals, tissues and cells with a reducedlevel of expression of functional αGT that concurrently express at leastone of the following in endothelium: 1) an immunomodulator including acomplement inhibitor (i.e. CD46, CD55, CD59, CR1 and the like) or animmunosuppressor (i.e. CTLA-4, B7 and the like) or 2) an anticoagulant(i.e. TFPI, hirudin, thrombomodulin, EPCR, CD39 and the like) are alsoincluded in this invention. In some embodiments, animals, tissue andcells are provided that have a reduced level of expression of functionalαGT and express both at least one immunomodulator and at least oneanticoagulant in endothelium. The complete or reduced level ofexpression of functional αGT may be achieved by any means known to oneof skill in the art. In one aspect of the present invention, porcineanimals are provided in which one allele of the αGT gene is inactivatedvia a genetic targeting event. In another aspect of the presentinvention, porcine animals are provided in which both alleles of the αGTgene are inactivated via a genetic targeting event. In one embodiment,the gene can be targeted via homologous recombination. In otherembodiments, the gene can be disrupted, i.e. a portion of the geneticcode can be altered, thereby affecting transcription and/or translationof that segment of the gene. For example, disruption of a gene can occurthrough substitution, deletion (“knock-out”) or insertion (“knock-in”)techniques. Additional genes for a desired protein or regulatorysequence that modulate transcription of an existing sequence can beinserted.

In embodiments of the present invention, the alleles of the αGT gene arerendered inactive, such that the resultant αGT enzyme can no longergenerate Gal on the cell surface. In one embodiment, the αGT gene can betranscribed into RNA, but not translated into protein. In anotherembodiment, the αGT gene can be transcribed in a truncated form. Such atruncated RNA can either not be translated or can be translated into anonfunctional protein. In an alternative embodiment, the αGT gene can beinactivated in such a way that no transcription of the gene occurs. In afurther embodiment, the αGT gene can be transcribed and then translatedinto a nonfunctional protein. In some embodiments, the expression ofactive αGT can be reduced by use of alternative methods, such as thosetargeting transcription or translation of the gene. For example, theexpression can be reduced by use of antisense RNA or siRNA targeting thenative αGT gene or an mRNA thereof. In other embodiments, site specificrecombinases are used to target a region of the genome forrecombination. Examples of such systems are the CRE-lox system and theFlp-Frt systems.

Pigs that possess two inactive alleles of the αGT gene are not naturallyoccurring. It was previously discovered that while attempting toknockout the second allele of the αGT gene through a genetic targetingevent, a point mutation was identified, which prevented the secondallele from producing functional αGT enzyme.

Thus, in another aspect of the present invention, the αGT gene can berendered inactive through at least one point mutation. In oneembodiment, one allele of the αGT gene can be rendered inactive throughat least one point mutation. In another embodiment, both alleles of theαGT gene can be rendered inactive through at least one point mutation.In one embodiment, this point mutation can occur via a genetic targetingevent. In another embodiment, this point mutation can be naturallyoccurring. In a further embodiment, mutations can be induced in the αGTgene via a mutagenic agent.

In one specific embodiment the point mutation can be a T-to-G mutationat the second base of exon 9 of the αGT gene. Pigs carrying a naturallyoccurring point mutation in the αGT gene allow for the production ofαGT-deficient pigs free of antibiotic-resistance genes and thus have thepotential to make a safer product for human use. In other embodiments,at least two, at least three, at least four, at least five, at least tenor at least twenty point mutations can exist to render the αGT geneinactive. In other embodiments, pigs are provided in which both allelesof the αGT gene contain point mutations that prevent any expression offunctional αGT enzyme. In a specific embodiment, pigs are provided thatcontain the T-to-G mutation at the second base of exon 9 in both allelesof the αGT gene.

Another aspect of the present invention provides a porcine animal, inwhich both alleles of the αGT gene are inactivated, whereby one alleleis inactivated by a genetic targeting event and the other allele isinactivated via a mutation. In one embodiment, a porcine animal isprovided, in which both alleles of the αGT gene are inactivated, wherebyone allele is inactivated by a genetic targeting event and the otherallele is inactivated due to presence of a T-to-G point mutation at thesecond base of exon 9. In a specific embodiment, a porcine animal isprovided, in which both alleles of the αGT gene are inactivated, wherebyone allele is inactivated via a targeting construct directed to Exon 9and the other allele is inactivated due to presence of a T-to-G pointmutation at the second base of exon 9.

Immunomodulators

Immunomodulators can be complement regulators and immunosuppressants.

(i) Complement Regulators

Complement is the collective term for a series of blood proteins and isa major effector mechanism of the immune system. Complement activationand its deposition on target structures can lead to directcomplement-mediated cell lysis or can lead indirectly to cell or tissuedestruction due to the generation of powerful modulators of inflammationand the recruitment and activation of immune effector cells. Complementactivation products that mediate tissue injury are generated at variouspoints in the complement pathway. Inappropriate complement activation onhost tissue plays an important role in the pathology of many autoimmuneand inflammatory diseases, and is also responsible for many diseasestates associated with bioincompatibility, e.g. post-cardiopulmonaryinflammation and transplant rejection. Complement deposition on hostcell membranes is prevented by complement inhibitory proteins expressedat the cell surface.

The complement system comprises a collection of about 30 proteins and isone of the major effector mechanisms of the immune system. Thecomplement cascade is activated principally via either the classical(usually antibody-dependent) or alternative (usuallyantibody-independent) pathways. Activation via either pathway leads tothe generation of C3 convertase, which is the central enzymatic complexof the cascade. C3 convertase cleaves serum C3 into C3a and C3b, thelatter of which binds covalently to the site of activation and leads tothe further generation of C3 convertase (amplification loop). Theactivation product C3b (and also C4b generated only via the classicalpathway) and its breakdown products are important opsonins and areinvolved in promoting cell-mediated lysis of target cells (by phagocytesand NK cells) as well as immune complex transport and solubilization.C3/C4 activation products and their receptors on various cells of theimmune system are also important in modulating the cellular immuneresponse. C3 convertases participate in the formation of C5 convertase,a complex that cleaves C5 to yield C5a and C5b. C5a has powerfulproinflammatory and chemotactic properties and can recruit and activateimmune effector cells. Formation of C5b initiates the terminalcomplement pathway resulting in the sequential assembly of complementproteins C6, C7, C8 and (C9)n to form the membrane attack complex (MACor C5b-9). Formation of MAC in a target cell membrane can result indirect cell lysis, but can also cause cell activation and theexpression/release of various inflammatory modulators.

There are two broad classes of membrane complement inhibitor inhibitorsof the complement activation pathway (inhibit C3 convertase formation),and inhibitors of the terminal complement pathway (inhibit MACformation). Membrane inhibitors of complement activation includecomplement receptor 1 (CR1), decay-accelerating factor (DAF or CD55) andmembrane cofactor protein (MCP or CD46). They all have a proteinstructure that consists of varying numbers of repeating units of about60-70 amino acids termed short consensus repeats (SCR) that are a commonfeature of C3/C4 binding proteins. Rodent homologues of human complementactivation inhibitors have been identified. The rodent protein Cr1 is awidely distributed inhibitor of complement activation that functionssimilar to both DAF and MCP. Rodents also express DAF and MCP, althoughCr1 appears to be functionally the most important regulator ofcomplement activation in rodents. Although there is no homolog of Cr1found in humans, the study of Cr1 and its use in animal models isclinically relevant.

Control of the terminal complement pathway and MAC formation in hostcell membranes occurs principally through the activity of CD59, a widelydistributed 20 kD glycoprotein attached to plasma membranes by aglycosylphosphatidylinositol (GPI) anchor. CD59 binds to C8 and C9 inthe assembling MAC and prevents membrane insertion.

Host cells are protected from their own complement by membrane-boundcomplement regulatory proteins like DAF, MCP and CD59. When an organ istransplanted into another species, natural antibodies in the recipientbind the endothelium of the donor organ and activate complement, therebyinitiating rapid rejection. It has previously been suggested that, incontrast to human cells, those of the pig are very susceptible to humancomplement, and it was thought that this was because pig cell-surfacecomplement regulatory proteins are ineffective against human complement.When an organ is transplanted into another species, natural antibodiesin the recipient bind the endothelium of the donor organ and activatecomplement, thereby initiating rapid rejection. Several strategies havebeen shown to prevent or delay rejection, including removal of IgMnatural antibodies and systemic decomplementation or inhibition ofcomplement using sCR1, heparin or C1 inhibitor.

An alternative approach to the problem of rejection is to express human,membrane-bound, complement-regulatory molecules in transgenic pigs.Transgenic pigs expressing decay acceleration factor DAF (CD55),membrane co-factor protein MCP (CD46) and membrane inhibitor of reactivelysis, MIRL (CD59) have been generated. (see Klymium et al. Mol ReprodDev (2010)77:209-221). These human inhibitors have been shown to beabundantly expressed on porcine vascular endothelium. Ex vivo perfusionof hearts from control animals with human blood causedcomplement-mediated destruction of the organ within minutes, whereashearts obtained from transgenic animals were refractory to complementand survived for hours.

The rationale for expressing human complement regulatory proteins in pigorgans to “humanize” them as outlined above is based on the assumptionthat endogenous pig regulatory proteins are inefficient at inhibitinghuman complement and thus will contribute little to organ survival inthe context of xenotransplantation. U.S. Pat. No. 7,462,466 to Morgan etal. describes the isolation and characterization of porcine analogues ofseveral of the human complement regulatory proteins (CRP). The studiesillustrated that pig organs expressing human complement regulatoryprotein molecules were resistant to complement damage not because theyexpressed human CRP molecules, but because they expressed greatlyincreased amounts of functional CRP molecules. Morgan et al. found thatincreased expression of porcine CRP could be equally effective inprotecting the donor organ from complement damage leading to hyperacuterejection as donor organs expressing human complement regulatoryproteins.

CD46 has been characterized as a protein with regulatory properties ableto protect the host cell against complement mediated attacks activatedvia both classical and alternative pathways (Barilla-LaBarca, M. L. etal., J. Immunol. 168, 6298-6304 (2002)). hCD46 may offer protectionagainst complement lysis during inflammation and humoral rejectionmediated by low levels of natural or induced anti-Gal or anti-nonGalantibodies. Transgenic pigs with the combination of GTKO and expressionof CD46 provided prolonged survival and function of xenograft hearts(pig-to baboon) for up to 8 months without any evidence of immunerejection (Mohiuddin et al., Abstract TTS-1383. Transplantation 2010; 90(suppl): 325).

In one embodiment of the present invention, animals, organs, tissues andcells are provided that express at least one complement regulator andeither lack any expression of functional αGT or express at least one ofthe following in endothelium: 1) an immunosuppressor (i.e. CTLA-4, B7and the like) or 2) an anticoagulant (i.e. TFPI, hirudin,thrombomodulin, EPCR, CD39 and the like).

In other embodiments, animals, organs, tissue and cells are providedthat express at least one complement regulator, lack any expression offunctional αGT and express at least one of the following inendothelium: 1) an immunosuppressor (i.e. CTLA-4, B7 and the like) or 2)an anticoagulant (i.e. TFPI, hirudin, thrombomodulin, EPCR, CD39 and thelike).

In still further embodiments, animals, organs, tissue and cells areprovided that express at least one complement regulator, lack anyexpression of functional αGT, express at least one immunosuppressor(i.e. CTLA-4, B7 and the like), and express at least one anticoagulant(i.e. TFPI, hirudin, thrombomodulin, EPCR, CD39 and the like) inendothelium. In some embodiments, the complement regulator may be acomplement inhibitor. In further embodiments, the complement inhibitormay be a membrane complement inhibitor. The membrane complementinhibitor may be either an inhibitor of the complement activationpathway (inhibit C3 convertase formation) or an inhibitor of theterminal complement pathway (inhibit MAC formation). Membrane inhibitorsof complement activation include complement receptor 1 (CR1),decay-accelerating factor (DAF or CD55), membrane cofactor protein (MCPor CD46) and the like. Membrane inhibitors of the terminal complementpathway may include CD59 and the like. In instances where complementregulators are expressed, two or more different complement regulatorsmay be expressed.

In some embodiments of the present invention, the complement regulatorsare human complement regulators. In other embodiments, the complementregulators are porcine complement regulators.

In a particular embodiment, the compliment inhibitor (e.g., CD46 or DAF)is expressed in every cell where it would normally be expressed. Inanother embodiment, the compliment inhibitor is expressed ubiquitously.

In one embodiment, the animals, organs, tissues or cells according tothe present invention, can be modified to transgenically express the oneor more complement regulators. The animals, organs, tissues or cells canbe modified to express a complement regulator peptide, a biologicallyactive fragment or derivative thereof. In one embodiment, the complementregulator peptide is the full length complement regulator. In a furtherembodiment, the complement regulator peptide can contain less than thefull length complement regulator protein.

Any human or porcine complement regulator sequences or biologicallyactive portion or fragment thereof known to one skilled in the art canbe according to the compositions and methods of the present invention.In additional embodiments, any consensus complement regulator peptidecan be used according to the present invention. In another embodiment,nucleic acid and/or peptide sequences at least 80%, 85%, 90% or 95%homologous to the complement regulator peptides and nucleotide sequencesdescribed herein. In further embodiments, any fragment or homologoussequence that exhibits similar activity as complement regulator can beused.

(ii) Immunosuppressants

An “immunosuppressant” transgene is capable of downregulating an immuneresponse. For any type of transplantation procedure, a balance betweenefficacy and toxicity is a key factor for its clinical acceptance. A

Biological agents that block key T cell costimulatory signals, inparticular the CD28 pathway, have potential to protect xenografts.Examples of agents that block the CD28 pathway include but are notlimited to soluble CTLA4 including mutant CTLA4 molecules.

T-cell activation is involved in the pathogenesis of transplantrejection. Activation of T-cells requires at least two sets of signalingevents. The first is initiated by the specific recognition through theT-cell receptor of an antigenic peptide combined with majorhistocompatibility complex (MHC) molecules on antigen presenting cells(APC5). The second set of signals is antigen nonspecific and isdelivered by T-cell costimulatory receptors interacting with theirligands on APCs. In the absence of costimulation, T-cell activation isimpaired or aborted, which may result in an antigen specificunresponsive state of clonal anergy, or in deletion by apoptotic death.Hence, the blockade of T-cell costimulation may provide an approach forsuppressing unwanted immune responses in an antigen specific mannerwhile preserving normal immune functions. (Dumont, F. J. 2004 Therapy 1,289-304).

Of several T cell costimulatory pathways identified to date, the mostprominent is the CD28 pathway. CD28, a cell surface molecule expressedon T-cells, and its counter receptors, the B7.1 (CD80) and B7.2 (CD86)molecules, present on dendritic cells, macrophages, and B-cells, havebeen characterized and identified as attractive targets for interruptingT-cell costimulatory signals. A second T-cell surface moleculehomologous to CD28 is known as cytoxic T-lymphocyte associated protein(CTLA4). CTLA4 is a cell surface signaling molecule, but contrary to theactions of CD28, CTLA4 negatively regulates T cell function. CTLA4 has20-fold higher affinity for the B7 ligands than CD28. The gene for humanCTLA4 was cloned in 1988 and chromosomally mapped in 1990 (Dariavach etal., Eur. J. Immunol. 18:1901-1905 (1988); Lafage-Pochitaloff et al.,Immunogenetics 31:198-201 (1990); U.S. Pat. No. 5,977,318).

The CD28/B7 pathway has become an attractive target for interrupting Tcell costimulatory signals. The design of a CD28/B7 inhibitor hasexploited the endogenous negative regulator of this system, CTLA4. ACTLA4-immunoglobulin (CTLA4-Ig) fusion protein has been studiedextensively as a means to inhibit T cell costimulation. A difficultbalance must be reached with any immunosuppressive therapy; one mustprovide enough suppression to overcome the disease or rejection, butexcessive immunosuppression will inhibit the entire immune system. Theimmunosuppressive activity of CTLA4-Ig has been demonstrated inpreclinical studies of animal models of organ transplantation andautoimmune disease. In certain embodiments, LEA29Y is substituted forCTLA4 when CTLA4 is embodied as the immunomodulator of the presentinvention.

Soluble CTLA4 has recently been tested in human patients with kidneyfailure, psoriasis and rheumatoid arthritis and has been formulated as adrug developed by Bristol-Myers Squibb (Abatacept, soluble CTLA4-Ig)that has been approved for the treatment of rheumatoid arthritis. Thisdrug is the first in the new class of selective T cell costimulationmodulators. Bristol-Myers Squibb is also conducting Phase II clinicaltrials with Belatacept (LEA29Y) for allograft kidney transplants. LEA29Yis a mutated form of CTLA4, which has been engineered to have a higheraffinity for the B7 receptors than wild-type CTLA4, fused toimmunoglobulin. Repligen Corporation is also conducting clinical trialswith its CTLA4-Ig for idiopathic thrombocytopenic purpura. U.S. Pat. No.5,730,403 entitled “Methods for protecting allogeneic islet transplantusing soluble CTLA4 mutant molecules”, describes the use of solubleCTLA4-Ig and CTLA4 mutant molecules to protect allogeneic islettransplants. Although CTLA-4 from one organism is able to bind to B7from another organism, the highest avidity is found for allogeneic B7.Thus, while soluble CTLA-4 from the donor organism can thus bind to bothrecipient B7 (on normal cells) and donor B7 (on xenotransplanted cells),it preferentially binds B7 on the xenograft. Thus in the embodiments ofthe invention comprising porcine animals or cells forxenotransplantation, porcine CTLA4 is typical. PCT Publication No. WO99/5 7266 by Imperial College describes a porcine CTLA4 sequence and theadministration of soluble CTLA4-Ig for xenotransplantation therapy.Vaughn A. et al., J Immunol (2000) 3175-3181, describes binding andfunction of soluble porcine CTLA4-Ig. Porcine CTLA4-Ig binds porcine(but not human) B7, blocking CD28 on recipient T cells and renderingthese local T cells anergic without causing global T cellimmunosuppression (see Mirenda et. al., Diabetes 54:1048-1055, 2005).

To date, much of the research on CTLA4-Ig as an immunosuppressive agenthas focused on administering soluble forms of CTLA4-Ig to the patient.Transgenic mice engineered to express CTLA4-Ig have been created andsubject to several lines of experimentation. Ronchese et al. examinedimmune system function generally after expression of CTLA4 in mice(Ronchese et al. J Exp Med (1994) 179: 809; Lane et al. J Exp Med.(1994) March 1; 179(3):819). Sutherland et al. (Transplantation. 200069(9):1806-12) described the protective effect of CTLA4-Ig secreted bytransgenic fetal pancreas allografts in mice to test the effects oftransgenically expressed CTLA4-Ig on allogenic islet transplantation.Lui et al. (J Immunol Methods 2003 277: 171-183) reported the productionof transgenic mice that expressed CTLA4-Ig under control of a mammaryspecific promoter to induce expression of soluble CTLA4-Ig in the milkof transgenic animals for use as a bioreactor.

PCT Publication No. WO 01/30966 by Alexion Pharmaceuticals Inc.describes chimeric DNA constructs containing the T cell inhibitor CTLA-4attached to the complement protein CD59, as well as transgenic porcinecells, tissues, and organs containing the same. PCT Publication No.WO2007035213 (Revivicor) describes transgenic porcine animals that havebeen genetically modified to express CTLA4-Ig.

Although the development of CTLA4-Ig expressing animals has beensuggested, these animals are severely immunocompromised. Recently, pigsproduced by Revivicor, Inc. expressing CTLA4-Ig ubiquitously using a CAG(ubiquitous) enhancer/promoter were found to have an immunocompromisedphenotype and were not viable in a typical husbandry environment (Phelpset al., 2009 Xenotransplantation. November-December; 16(6):477-85.Therefore there is a need to express such immunosuppressant transgenesin a tissue specific manner, such as in the endothelium of a xenograft,where high but localized levels of protein expression are possible,without any resulting phenotypic problems in the transgenic animal.

Additional immunomodulators, and in particular immunosuppressors can beexpressed in the animals, tissues or cells. For example, genes whichhave been inactivated in mice to produce an immuno compromisedphenotype, can be cloned and disrupted by gene targeting in pigs. Somegenes which have been targeted in mice and may be targeted to produceimmuno compromised pigs include beta 2-microglobulin (MHC class Ideficiency, Koller et al., Science, 248:1227-1230), TCR alpha, TCR beta(Mombaerts et al., Nature, 360:225-231), RAG-1 and RAG-2 (Mombaerts etal., (1992) Cell 68, 869-877, Shinkai, et al., (1992) Cell 68, 855-867,U.S. Pat. No. 5,859,307).

In one embodiment, the animals, organs, tissues, or cells according tothe present invention, can be modified to transgenically express acytoxic T-lymphocyte associated protein 4-immunoglobin (CTLA4). Theanimals or cells can be modified to express CTLA4 peptide or abiologically active fragment (e.g., extracellular domain, truncated formof the peptide in which at least the transmembrane domain has beenremoved) or derivative thereof. The peptide may be, e.g., human orporcine. The CTLA4 peptide can be mutated. Mutated peptides may havehigher affinity than wildtype for porcine and/or human B7 molecules. Inone specific embodiment, the mutated CTLA4 can be CTLA4 (Glu104, Tyr29).The CTLA4 peptide can be modified such that it is expressedintracellularly. Other modifications of the CTLA4 peptide includeaddition of an endoplasmic reticulum retention signal to the N or Cterminus. The endoplasmic reticulum retention signal may be, e.g., thesequence KDEL. The CTLA4 peptide can be fused to a peptide dimerizationdomain or an immunoglobulin (Ig) molecule. The CTLA4 fusion peptides caninclude a linker sequence that can join the two peptides. In anotherembodiment, animals lacking expression of functional immunoglobulin,produced according to the present invention, can be administered a CTLA4peptide or a variant thereof (pCTLA4-Ig, or hCTLA4-Ig(Abatacept/Orencia, or Belatacept) as a drug to suppress their T-cellresponse.

In one embodiment, the CTLA4 peptide is the full length CTLA4. In afurther embodiment, the CTLA4 peptide can contain less than the fulllength CTLA4 protein. In one embodiment, the CTLA4 peptide can containthe extracellular domain of a CTLA-4 peptide. In a particularembodiment, the CTLA4 peptide is the extracellular domain of CTLA4. Instill further embodiments, the present invention provides mutated formsof CTLA4. In one embodiment, the mutated form of CTLA4 can have higheraffinity than wild type for porcine and/or human B7. In one specificembodiment, the mutated CTLA4 can be human CTLA4 (Glu104, Tyr29).

In one embodiment, the CTLA4 can be a truncated form of CTLA4, in whichat least the transmembrane domain of the protein has been removed. Inanother embodiment, the CTLA4 peptide can be modified such that it isexpressed intracellularly. In one embodiment, a golgi retention signalcan be added to the N or C terminus of the CTLA4 peptide. In oneembodiment, the golgi retention signal can be the sequence KDEL, whichcan be added to the C or N terminal of the CTLA4 peptide. In furtherembodiments, the CTLA4 peptide can be fused to a peptide dimerizationdomain. In one embodiment, the CTLA4 peptide can be fused to animmunoglobulin (Ig). In another embodiment, the CTLA4 fusion peptidescan include a linker sequence that can join the two peptides.

Any human CTLA4 sequences or biologically active portion or fragmentthereof known to one skilled in the art can be according to thecompositions and methods of the present invention. Non-limiting examplesinclude, but are not limited to the following Genbank accession numbersthat describe human CTLA4 sequences: NM005214.2; BC074893.2; BC074842.2;AF414120.1; AF414120; AY402333; AY209009.1; BC070162.1; BC069566.1;L15006.1; AF486806.1; AC010138.6; AJ535718.1; AF225900.1; AF225900;AF411058.1; M37243.1; U90273.1; and/or AF316875.1. Further nucleotidesequences encoding CTLA4 peptides can be selected from those including,but not limited to the following Genbank accession numbers from the ESTdatabase: CD639535.1; A1733018.1; BM997840.1; BG536887.1; BG236211.1;BG058720.1; A1860i99.1; AW207094.1; AA210929.1; A1791416.1; BX113243.1;AW515943.1; BE837454.1; AA210902.1; BF329809.1; A1819438.1; BE837501.1;BE837537.1; and/or AA873138.1.

In additional embodiments, any consensus CTLA4 peptide can be usedaccording to the present invention. In another embodiment, nucleic acidand/or peptide sequences at least 80%, 85%, 90% or 95% homologous to thenative CTLA4 peptides and nucleotide sequences. In further embodiments,any fragment or homologous sequence that exhibits similar activity asCTLA4 can be used.

In other embodiments, the amino acid sequence which exhibits T cellinhibitory activity can be amino acids 38 to 162 of the porcine CTLA4sequence or amino acids 38 to 161 of the human CTLA4 sequence (see, forexample, PCT Publication No. WO 01/30966). In one embodiment, theportion used should have at least about 25% and preferably at leastabout 50% of the activity of the parent molecule.

In other embodiments, the CTLA4 nucleic acids and peptides of thepresent invention can be fused to immunoglobulin genes and molecules orfragments or regions thereof. Reference to the CTLA4 sequences of thepresent invention include those sequences fused to immunoglobulins.

In one embodiment, the Ig can be a human Ig. In another embodiment, theIg can be IgG, in particular, IgG1. In another embodiment, the Ig can bethe constant region of IgG. In a particular embodiment, the constantregion can be the Cγ1 chain of IgG1. In one particular embodiment of thepresent invention, the extracellular domain of porcine CTLA4 can befused to human Cγ1 Ig. In another particular embodiment, theextracellular domain of human CTLA4 can be fused to IgG1 or IgG4. In afurther particular embodiment, the extracellular domain of mutated CTLA4(Glu 104, Tyr 29) can be fused to IgG1.

(iii) Other Immunomodulators

Other immunodulators that can be used include class II transactivators(CIITA) and mutants thereof, PDL1, PDL2, tumor necrosis factor-α-relatedapoptosis-inducing ligand (TRAIL), Fas ligand (FasL, CD95L)integrin-associated protein (CD47), HLA-E, HLA-DP, HLA-DQ, or HLA-DR.

(a) CIITA: The class II transactivator (CIITA) is a bi- ormultifunctional domain protein that acts as a transcriptional activatorand plays a critical role in the expression of MHC class II genes. Ithas been previously demonstrated that a mutated form of the human CIITAgene, coding for a protein lacking the amino terminal 151 amino acids,acts as a potent dominant-negative suppressor of HLA class II expression(Yun et al., Int Immunol. 1997 October; 9(10):1545-53). Porcine MHCclass II antigens are potent stimulators of direct T-cell recognition byhuman CD4+ T cells and are, therefore, likely to play an important rolein the rejection responses to transgenic pig donors in clinicalxenotransplantation. It was reported that one mutated human CIITAconstruct was effective in pig cells, markedly suppressingIFN[gamma]-induced as well as constitutive porcine MHC class IIexpression. Moreover, stably transfected porcine vascular endothelialcell lines carrying mutated human CIITA constructs failed to stimulatedirect T-cell xenorecognition by purified human CD4+ T cells (Yun etal., Transplantation. 2000 Mar. 15; 69(5):940-4). Organs, tissues andcells from CIITA-DN transgenic animals could induce a much reducedT-cell rejection responses in human recipients. In combination withother transgenes, transgenic expression of a mutated CIITA might enablelong-term xenograft survival with clinically acceptable levels ofimmunosuppression. In one embodiment, a human CIITA can be used. Inparticular, a human CIITA-DN. In another embodiment, a porcine CIITA canbe used. In particular, a porcine CIITA-DN.

(b) PDL1, PDL2: Typical costimulatory molecules for T-cell activationare CD80/86 or CD40. In addition to these positive costimulatorypathways over the past several years, new costimulatory pathways thatmediate negative signals and are important for the regulation of T-cellactivation have been found. One of these newer pathways is the pathwayconsisting of Programmed death 1 (PD-1) receptor and its ligands, PD-L1and PD-L2. The PD-1 receptor is not expressed in resting cells but isupregulated after T and B cell activation. PD-1 contains a cytoplasmicimmunoreceptor tyrosine-based switch motif and binding of PD-L1 or PD-L2to PD-1 leads to inhibitory signals in T cells. Recent data suggest thatPD1/PDLigand pathways may play a role in the control of T-cell subsetsexhibiting regulatory activity. In mice, PD-1 signals have been shown tobe required for the suppressive activity of regulatory T cells (Treg)and the generation of adaptive Treg. These observations suggest thatPD-1/PDLig and interactions do not only inhibit T-cell responses but mayalso provoke immunoregulation. Several lines of evidence demonstratethat PD-1/PDLigand pathways can control engraftment and rejection ofallografts implying that these molecules are interesting targets forimmunomodulation after organ transplantation. Indeed, prolongation ofallograft survival could be obtained by PDL1Ig gene transfer to donorhearts in a rat transplantation model. Moreover, enhancing PD-1signaling by injection of PD-L 1Ig has also been reported to protectgrafts from rejection in mice. Recent data also show that overexpressionof PD-L1IG on islet grafts in mice can partially prolong islet graftsurvival. Transgenic expression of human PD-L1 or PD-L2 in pig cells andtissues should reduce early human anti-pig T-cell responses initiatedvia the direct route of sensitization (Plege et al., Transplantation.2009 Apr. 15; 87(7):975-82). By the induction of Treg it might also bepossible to control T cells sensitized to the xenograft through theindirect route that is required to achieve long-lasting tolerance.

(c) TRAIL/Fas L: Expression of apoptosis inducing ligands, such as Fasligand (FasL, CD95L) or tumor necrosis factor-α-relatedapoptosis-inducing ligand (TRAIL, Apo-2L) may eliminate T cellsattacking a xenograft. TRAIL is a type II membrane protein with anextracellular domain homologous to that of other tumor necrosis factorfamily members showing the highest amino acid identity to FasL (28%).TRAIL exerts its apoptosis-inducing action preferentially on tumorcells. In normal cells, binding of TRAIL receptors does not lead to celldeath. Recent studies have shown that the cytotoxic effects of immunecells, including T cells, natural killer cells, macrophages, anddendritic cells, are mediated at least partly by TRAIL. Expression ofhuman TRAIL in transgenic pigs may provide a reasonable strategy forprotecting pig tissues against cell-mediated rejection afterxenotransplantation to primates. Stable expression of human TRAIL hasbeen achieved in transgenic pigs and TRAIL expressed has been shown tobe biologically functional in vitro (Klose et al., Transplantation. 2005Jul. 27; 80(2):222-30).

(d) CD47: CD47, known as integrin-associated protein, is a ubiquitouslyexpressed 50 kDa cell surface glycoprotein that serves as a ligand forsignal regulatory protein (SIRP)α (also known as CD172a, SHPS-1), animmune inhibitory receptor on macrophages. CD47 and SIRPα constitute acell-cell communication system (the CD47-SIRPα system) that playsimportant roles in a variety of cellular processes including cellmigration, adhesion of B cells, and T cell activation. In addition, theCD47-SIRPα system is implicated in negative regulation of phagocytosisby macrophages. CD47 on the surface of several cell types (i.e.,erythrocytes, platelets, or leukocytes) can protect against phagocytosisby macrophages by binding to the inhibitory macrophage receptor SIRPα.The role of CD47-SIRPα interactions in the recognition of self andinhibition of phagocytosis has been illustrated by the observation thatprimary, wild-type mouse macrophages rapidly phagocytose unopsonizedRBCs obtained from CD47-deficient mice but not those from wild-typemice. It has also been reported that through its SIRPα receptors, CD47inhibits both Fcγ and complement receptor-mediated phagocytosis. It hasbeen demonstrated that porcine CD47 does not induce SIRPα tyrosinephosphorylation in human macrophage-like cell line, and soluble humanCD47-Fc fusion protein inhibits the phagocytic activity of humanmacrophages toward porcine cells. It was also indicated thatmanipulation of porcine cells for expression of human CD47 radicallyreduces the susceptibility of the cells to phagocytosis by humanmacrophages (Ide et al., Proc Natl Acad Sci USA. 2007 Mar. 20;104(12):5062-6). Expression of human CD47 on porcine cells could provideinhibitory signaling to SIRPα on human macrophages, providing anapproach to preventing macrophage-mediated xenograft rejection.

(e) NK Cell Response. HLA-E/Beta 2 Microglobulin and HLA-DP, HLA-DQ,HLA-DR:

Human natural killer (NK) cells represent a potential hurdle tosuccessful pig-to-human xenotransplantation because they infiltrate pigorgans perfused with human blood ex vivo and lyse porcine cells in vitroboth directly and, in the presence of human serum, by antibody-dependentcell-mediated cytotoxicity. NK cell autoreactivity is prevented by theexpression of major histocompatibility complex (MHC) class I ligands ofinhibitory NK receptors on normal autologous cells. The inhibitoryreceptor CD94/NKG2A that is expressed on a majority of activated humanNK cells binds specifically to human leukocyte antigen (HLA)-E. Thenonclassical human MHC molecule HLA-E is a potent inhibitory ligand forCD94/NKG2A-bearing NK cells and, unlike classical MHC molecules, doesnot induce allogeneic T-cell responses. HLA-E is assembled in theendoplasmic reticulum and transported to the cell surface as a stabletrimeric complex consisting of the HLA-E heavy chain, β2-microglobulin(β 2 m), and a peptide derived from the leader sequence of some MHCclass 1 molecules. The expression of HLA-E has been shown to providepartial protection against xenogeneic human NK cell cytotoxicity (Weisset al., Transplantation. 2009 Jan. 15; 87(1):35-43). Transgenicexpression of HLA-E on pig organs has the potential to substantiallyalleviate human NK cell-mediated rejection of porcine xenografts withoutthe risk of allogeneic responses. In addition, transgenic pigs carryingother HLA genes have been successfully generated with the goal of“humanizing” porcine organs, tissues, and cells (Huang et al.,Proteomics. 2006 November; 6(21):5815-25, see also U.S. Pat. No.6,639,122).

Anticoagulants

In certain embodiments of the present invention, anticoagulanttransgenes can be introduced into porcine animals. Such transgenes canbe expressed specifically in the porcine endothelium. In one embodimentof the current invention, the Tie-2 enhancer and promoter can be used.The Tie-2 enhancer and promoter have been shown to provide uniformvascular-endothelial-cell-specific gene expression in embryonic andadult transgenic mice (Schlaeger et al., 1997 Proc Natl Acad Sci. April1; 94(7):3058-63). In one example, the Tie-2 promoter and enhancer wasutilized to construct a vector for driving expression of ananticoagulant, locally and specifically, in the endothelium of theresulting transgenic animals. In another embodiment of the currentinvention, the porcine ICAM-2 promoter, and portions of its first introncontaining enhancer activity (also termed the “ICAM-2 enhancer” hereincan be used. In one example, the porcine ICAM-2 promoter, and portionsof its first intron containing enhancer activity (also termed the“ICAM-2 enhancer” herein) (Godwin et al., 2006. Xenotransplantation.November; 13(6):514-21) was utilized to construct a second vector fordriving expression of an anticoagulant, locally and specifically, in theendothelium of the resulting transgenic animals.

In certain embodiments of the present invention, Tissue factor pathwayinhibitor (TFPI) can be used as the anticoagulant, TFPI is asingle-chain polypeptide which can reversibly inhibit Factor Xa (Xa) andThrombin (Factor IIa) and thus inhibits TF dependent coagulation. For areview of TFPI, please see Crawley and Lane (Arterioscler Thromb VascBiol. 2008, 28(2):233-42). Dorling and colleagues generated transgenicmice expressing a fusion protein consisting of the three Kunitz domainsof human TFPI linked to the transmembrane/cytoplasmic domains of humanCD4, with a P-selectin tail for targeting to Weibel-Palade intracellularstorage granules (Chen D, et al. Am J Transplant 2004; 4: 1958-1963.).The resulting activation-dependent display of TFPI on the endotheliumwas sufficient to completely inhibit thrombosis-mediated acute humoralrejection of mouse cardiac xenografts by cyclosporine-treated rats.There was also a suggestion that effective regulation of coagulation mayprevent chronic rejection. Similar results were obtained with transgenicmouse hearts expressing a hirudin/CD4/P-selectin fusion protein,indicating that inhibition of thrombin generation or activity was thekey to protection in this model.

In certain embodiments, hirudin can be used as the anticoagulant of thepresent invention. Hirudin is a naturally occurring peptide in thesalivary glands of medicinal leeches (such as Hirudo medicinalis) and isa potent inhibitor of thrombin. Dorling and coworkers (Chen et al., JTransplant. 2004 December; 4(12):1958-63) also generated transgenic miceexpressing membrane-tethered hirudin fusion proteins, and transplantedtheir hearts into rats (mouse-rat Xeno-Tx). In contrast to controlnon-transgenic mouse hearts, which were all rejected within 3 days, 100%of the organs from both strains of transgenic mice were completelyresistant to humoral rejection and survived for more than 100 days whenT-cell-mediated rejection was inhibited by administration ofcyclosporine A. Riesbeck et al., (Circulation. 1998 Dec. 15;98(24):2744-52) also explored the expression of hirudin fusion proteinsin mammalian cells as a strategy for prevention of intravascularthrombosis. Expression in cells reduced local thrombin levels andinhibited fibrin formation. Therefore, hirudin is another anticoagulanttransgene of interest for preventing the thrombotic effects present inxenotransplantation.

In other certain embodiments, thrombomodulin can be used as theanticoagulant of the present invention. Thrombomodulin (TM) functions asa cofactor in the thrombin-induced activation of protein C in theanticoagulant pathway by forming a 1:1 stoichiometric complex withthrombin. Endothelial cell protein C receptor (EPCR) is anN-glycosylated type I membrane protein that enhances the activation ofprotein C. The role of these proteins in the protein C anticoagulantsystem is reviewed by Van de Wouwer et al., Arterioscler Thromb VascBiol. 2004 August; 24(8):1374-83. Expression of these and otheranticoagulant transgenes has been explored by various groups topotentially address the coagulation barriers to xenotransplantation(reviewed by Cowan and D'Apice, Cur Opin Organ Transplant. 2008 April;13(2):178-83). Esmon and coworkers (Li et al., J Thromb Haemost. 2005July; 3(7):1351-9 over-expressed EPCR on the endothelium of transgenicmice and showed that such expression protected the mice from thromboticchallenge. Iino et al., (J Thromb Haemost. 2004 May; 2(5):833-4),suggested ex-vivo over expression of TM in donor islets via gene therapyas a means to prevent thrombotic complications in islet transplantation.

In certain embodiments, CD39 can be used as the anticoagulant of thepresent invention. CD39 is a major vascular nucleoside triphosphatediphosphohydrolase (NTPDase), and converts ATP, and ADP to AMP andultimately adenosine. Extracellular adenosine plays an important role inthrombosis and inflammation, and thus has been studied for itsbeneficial role in transplantation (reviewed by Robson et al. SeminThromb Hemost. 2005 April; 31(2):217-33). Recent studies have shown thatCD39 has a major effect in reducing the inflammatory response (Beldi etal., Front Biosci, 2008, 13:2588-2603). Transgenic mice expressing hCD39exhibited impaired platelet aggregation, prolonged bleeding times, andresistance to systemic thromboembolism in a heart transplant model(Dwyer et al., J Clin Invest. 2004 May; 113(10):1440-6). They were alsoshown to express CD39 on pancreatic islets and when incubated with humanblood, these islets significantly delayed clotting time compared to wildtype islets (Dwyer et al., Transplantation. 2006 Aug. 15; 82(3):428-32).Preliminary efforts at expressing hCD39 at high levels from aconstitutive promoter system in transgenic pigs, showed high post-natallethality (Revivicor, Inc., unpublished data). Thus there is a need toexpress anticoagulant transgenes in pigs in a manner that does notcompromise the animal's well being, yet still provides adequate levelsof expression for utility in clinical xenotransplantation.

Cytoprotective Transgenes

The present invention includes cytoprotective transgenes(“cytoprotectants’). Cytoprotective transgenes are considered to includeanti-apoptotics, anti-oxidants and anti-inflammatories. Examplesinclude:

(a) A20: In certain embodiments, A20 can be used as the cytoprotectivetransgene of the present invention. A20 provides anti-inflammatory andanti-apoptotic activity. Vascularized transplanted organs may beprotected against endothelial cell activation and cellular damage byanti-inflammatory, anticoagulant and/or anti-apoptotic molecules. Amonggenes with great potential for modulation of acute vascular rejection(AVR) is the human A20 gene (hA20) that was first identified as a tumornecrosis factor (TNF)-α inducible factor in human umbilical veinendothelial cells. Human A20 has a double cytoprotective function byprotecting endothelial cells from TNF-mediated apoptosis andinflammation, via blockade of several caspases, and the transcriptionfactor nuclear factor-KB, respectively. Viable A20 transgenic pigletshave been produced and in these animals expression of hA20 wasrestricted to skeletal muscle, heart and PAECs which were protectedagainst TNF mediated apoptosis by hA20 expression and at least partlyagainst CD95(Fas)L-mediated cell death. In addition, cardiomyocytes fromhA20-transgenic-cloned pigs were partially protected against cardiacinsults (Oropeza et al., Xenotransplantation. 2009 November;16(6):522-34).

(b) HO-1: In certain embodiments, HO can be used as the cytoprotectivetransgene of the present invention. HO provides anti-inflammatory,anti-apoptotic, and anti-oxidant activity. Heme oxygenases (HOs),rate-limiting enzymes in heme catabolism, also named HSP32, belong tomembers of heat shock proteins, wherein the heme ring is cleaved intoferrous iron, carbon monoxide (CO) and biliverdin that is then convertedto bilirubin by biliverdin reductase. Three isoforms of HOs, includingHO-1, HO-2 and HO-3, have been cloned. The expression of HO-1 is highlyinducible, whereas HO-2 and HO-3 are constitutively expressed (Maines MD et al., Annual Review of Pharmacology & Toxicology 1997; 37:517-554,and Choi A M et al., American Journal of Respiratory Cell & MolecularBiology 1996; 15:9-19). An analysis of HO-1−/− mice suggests that thegene encoding HO-1 regulates iron homeostasis and acts as acytoprotective gene having potent antioxidant, anti-inflammatory andanti-apoptotic effects (Poss K D et al., Proceedings of the NationalAcademy of Sciences of the United States of America 1997;94:10925-10930, Poss K D et al., Proceedings of the National Academy ofSciences of the United States of America 1997; 94:10919-10924, andSoares M P et al., Nature Medicine 1998; 4:1073-1077). Similar findingswere recently described in a case report of HO-1 deficiency in humans(Yachie A et al., Journal of Clinical Investigation 1999; 103:129-135).The molecular mechanisms responsible for the cytoprotective effects ofHO-1, including anti-inflammation, anti-oxidation and anti-apoptosis,are mediated by its' reaction products. HO-1 expression can be modulatedin vitro and in vivo by protoporphyrins with different metals. Cobaltprotoporphyrins (CoPP) and iron protoporphyrins (FePP) can up-regulatethe expression of HO-1. In contrast, tin protoporphyrins (SnPP) and zincprotoporphyrins (ZnPP) inhibit the activity of HO-1 at the proteinlevel. Recently, it has been proved that the expression of HO-1suppresses the rejection of mouse-to-rat cardiac transplants (Sato K etal., J. Immunol. 2001; 166:4185-4194), protects islet cells fromapoptosis, and improves the in vivo function of islet cells aftertransplantation (Pileggi A et al., Diabetes 2001; 50: 1983-1991). It hasalso been proved that administration of HO-1 by gene transfer providesprotection against hyperoxia-induced lung injury (Otterbein L E et al.,J Clin Invest 1999; 103: 1047-1054), upregulation of HO-1 protectsgenetically fat Zucker rat livers from ischemia/reperfusion injury(Amersi F et al., J Clin Invest 1999; 104: 1631-1639), and ablation orexpression of HO-1 gene modulates cisplatin-induced renal tubularapoptosis (Shiraishi F et al., Am J Physiol Renal Physiol 2000;278:F726-F736). In transgenic animal models, it was shown thatover-expression of HO-1 prevents the pulmonary inflammatory and vascularresponses to hypoxia (Minamino T et al., Proc. Natl. Acad. Sci. USA2001; 98:8798-8803) and protects heart against ischemia and reperfusioninjury (Yet S F, et al., Cir Res 2001; 89:168-173). Pigs carrying a HO-1transgene have been produced however clinical effects related to theiruse in xenotransplantation were not reported (U.S. Pat. No. 7,378,569).

(c) FAT-1: In certain embodiments, FAT-1 can be used as thecytoprotective transgene of the present invention. FAT-1 providesanti-inflammatory activity. Polyunsaturated fatty acids (PUFAs) play arole in inhibiting (n-3 class) inflammation. Mammalian cells are devoidof desaturase that converts n-6 to n-3 PUFAs. Consequently, essentialn-3 fatty acids must be supplied with the diet. Unlike mammals, however,the free-living nematode Caenorhabditis elegans expresses a n-3 fattyacid desaturase that introduces a double bond into n-6-fatty acids atthe n-3 position of the hydrocarbon chains to form n-3 PUFAs. Transgenicmice have been generated that express the C. elegans fat-1 gene and,consequently, are able to efficiently convert dietary PUFAs of the 6series to PUFAs of 3-series, such as EPA (20:5 n-3) and DHA (22-6 n-3).(Kang et al., Nature. 2004 Feb. 5; 427(6974):504). Another groupproduced a transgenic mouse model wherein the codons of fat-1 cDNA werefurther optimized for efficient translation in mammalian systems;endogenous production of n-3 PUFAs was achieved through overexpressing aC. elegans n-3 fatty acid desaturase gene, mfat-1. This group showedthat cellular increase of n-3 PUFAs and reduction of n-6 PUFAs throughtransgenic expression of mfat-1 enhanced glucose-, amino acid-, andGLP-1-stimulated insulin secretion in isolated pancreatic islets of themice, and rendered the islets strongly resistant to cytokine-inducedcell death (Wei et al., Diabetes. 2010 February; 59(2):471-8).

(d) Soluble TNF-alpha receptor (sTNFR1): In certain embodiments, sTNFR1can be used as the cytoprotective transgene of the present invention.Tumor necrosis factor (TNF, cachexin or cachectin and formally known astumor necrosis factor-alpha) is a cytokine involved in systemicinflammation and is a member of a group of cytokines that stimulate theacute phase reaction. The primary role of TNF is in the regulation ofimmune cells. TNF is able to induce apoptotic cell death, to induceinflammation. Soluble TNF-alpha receptor 1 (sTNFR1) is an extracellulardomain of TNFR1 and an antagonist to TNF-alpha (Su et al., 1998.Arthritis Rheum. 41, 139-149). Transgenic expression of sTNFR1 inxenografts may have beneficial anti-inflammatory effects.

In other certain embodiments, SOD can be used as the cytoprotectivetransgenes of the present invention. In other embodiments, catalase canbe used as the cytoprotective transgenes of the present invention. Othercytoprotectives with relevant anti-oxidant properties include, withoutlimitation, SOD and catalase. Oxygen is the essential molecule for allaerobic organisms, and plays predominant role in ATP generation, namely,oxidative phosphorylation. During this process, reactive oxygen species(ROS) including superoxide anion (O(2)(−)) and hydrogen peroxide(H(2)O(2)) are produced as by-products. In man, an antioxidant defensesystem balances the generation of ROS. Superoxide dismutase (SOD) andcatalase are two enzymes with anti-oxidant properties. SOD catalyses thedismutation of superoxide radicals to hydrogen peroxide, the latterbeing converted to water by catalase and glutathione peroxidase.Cellular damage resulting from generation of ROS can occur in atransplant setting. Therefore there is an interest in expressinganti-oxidant genes ex vivo or transgenically in donor tissues. Ex vivogene transfer of EC-SOD and catalase were anti-inflammatory in a ratmodel of antigen induced arthritis (Dai et al., Gene Ther. 2003 April;10(7):550-8). In addition, delivery of EC-SOD and/or catalase genesthrough the portal vein markedly attenuated hepatic I/R injury in amouse model (He et al., Liver Transpl. 2006 December; 12(12):1869-79).Moreover, certain anticoagulants also provide anti-inflammatory activityincluding thrombomodulin, EPCR and CD39.

Production of Genetically Modified Animals

Genetically modified animals can be produced by any method known to oneof skill in the art including, but not limited to, selective breeding,nuclear transfer, introduction of DNA into oocytes, sperm, zygotes, orblastomeres, or via the use of embryonic stem cells.

In some embodiments, genetic modifications may be identified in animalsthat are then bred together to form a herd of animals with a desired setof genetic modifications (or a single genetic modification). Theseprogeny may be further bred to produce different or the same set ofgenetic modifications (or single genetic modification) in their progeny.This cycle of breeding for animals with desired genetic modification(s)may continue for as long as one desires. “Herd” in this context maycomprise multiple generations of animals produced over time with thesame or different genetic modification(s). “Herd” may also refer to asingle generation of animals with the same or different geneticmodification(s).

Cells useful for genetic modification (via, for example, but not limitedto, homologous recombination) include, by way of example, epithelialcells, neural cells, epidermal cells, keratinocytes, hematopoieticcells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes),erythrocytes, macrophages, monocytes, mononuclear cells, fibroblasts,cardiac muscle cells, and other muscle cells, etc. Moreover, the cellsused for producing the genetically modified animal (via, for example,but not limited to, nuclear transfer) can be obtained from differentorgans, e.g., skin, lung, pancreas, liver, stomach, intestine, heart,reproductive organs, bladder, kidney, urethra and other urinary organs,etc. Cells can be obtained from any cell or organ of the body, includingall somatic or germ cells.

Additionally, animal cells that can be genetically modified can beobtained from a variety of different organs and tissues such as, but notlimited to, skin, mesenchyme, lung, pancreas, heart, intestine, stomach,bladder, blood vessels, kidney, urethra, reproductive organs, and adisaggregated preparation of a whole or part of an embryo, fetus, oradult animal. In one embodiment of the invention, cells can be selectedfrom the group consisting of, but not limited to, epithelial cells,fibroblast cells, neural cells, keratinocytes, hematopoietic cells,melanocytes, chondrocytes, lymphocytes (B and T), macrophages,monocytes, mononuclear cells, cardiac muscle cells, other muscle cells,granulosa cells, cumulus cells, epidermal cells, endothelial cells,Islets of Langerhans cells, blood cells, blood precursor cells, bonecells, bone precursor cells, neuronal stem cells, primordial stem cells,adult stem cells, mesenchymal stem cells, hepatocytes, keratinocytes,umbilical vein endothelial cells, aortic endothelial cells,microvascular endothelial cells, fibroblasts, liver stellate cells,aortic smooth muscle cells, cardiac myocytes, neurons, Kupffer cells,smooth muscle cells, Schwann cells, and epithelial cells, erythrocytes,platelets, neutrophils, lymphocytes, monocytes, eosinophils, basophils,adipocytes, chondrocytes, pancreatic islet cells, thyroid cells,parathyroid cells, parotid cells, tumor cells, glial cells, astrocytes,red blood cells, white blood cells, macrophages, epithelial cells,somatic cells, pituitary cells, adrenal cells, hair cells, bladdercells, kidney cells, retinal cells, rod cells, cone cells, heart cells,pacemaker cells, spleen cells, antigen presenting cells, memory cells, Tcells, B-cells, plasma cells, muscle cells, ovarian cells, uterinecells, prostate cells, vaginal epithelial cells, sperm cells, testicularcells, germ cells, egg cells, leydig cells, peritubular cells, sertolicells, lutein cells, cervical cells, endometrial cells, mammary cells,follicle cells, mucous cells, ciliated cells, nonkeratinized epithelialcells, keratinized epithelial cells, lung cells, goblet cells, columnarepithelial cells, squamous epithelial cells, osteocytes, osteoblasts,and osteoclasts. In one alternative embodiment, embryonic stem cells canbe used. An embryonic stem cell line can be employed or embryonic stemcells can be obtained freshly from a host, such as a porcine animal. Thecells can be grown on an appropriate fibroblast-feeder layer or grown inthe presence of leukemia inhibiting factor (LIF).

Embryonic stem cells are a preferred germ cell type, an embryonic stemcell line can be employed or embryonic stem cells can be obtainedfreshly from a host, such as a porcine animal. The cells can be grown onan appropriate fibroblast-feeder layer or grown in the presence ofleukemia inhibiting factor (LIF).

Cells of particular interest include, among other lineages, stem cells,e.g. hematopoietic stem cells, embryonic stem cells, mesenchymal stemcells, etc., the islets of Langerhans, adrenal medulla cells which cansecrete dopamine, osteoblasts, osteoclasts, epithelial cells,endothelial cells, leukocytes, e.g. B- and T-lymphocytes, myelomonocyticcells, etc., neurons, glial cells, ganglion cells, retinal cells, livercells, e.g. hepatocytes, bone marrow cells, keratinocytes, hair folliclecells, and myoblast (muscle) cells.

In a particular embodiment, the cells can be fibroblasts orfibroblast-like cells having a morphology or a phenotype that is notdistinguishable from fibroblasts, or a lifespan before senescense of atleast 10 or at least 12 or at least 14 or at least 18 or at least 20days, or a lifespan sufficient to allow homologous recombination andnuclear transfer of a non-senescent nucleus; in one specific embodiment,the cells can be fetal fibroblasts. Fibroblast cells are a suitablesomatic cell type because they can be obtained from developing fetusesand adult animals in large quantities. These cells can be easilypropagated in vitro with a rapid doubling time and can be clonallypropagated for use in gene targeting procedures. The cells to be usedcan be from a fetal animal, or can be neonatal or from an adult animalin origin. The cells can be mature or immature and either differentiatedor non-differentiated.

Homologous Recombination

Homologous recombination permits site-specific modifications inendogenous genes and thus novel alterations can be engineered into thegenome. A primary step in homologous recombination is DNA strandexchange, which involves a pairing of a DNA duplex with at least one DNAstrand containing a complementary sequence to form an intermediaterecombination structure containing heteroduplex DNA (see, for exampleRadding, C. M. (1982) Ann. Rev. Genet. 16: 405; U.S. Pat. No.4,888,274). The heteroduplex DNA can take several forms, including athree DNA strand containing triplex form wherein a single complementarystrand invades the DNA duplex (Hsieh et al. (1990) Genes and Development4: 1951; Rao et al., (1991) PNAS 88:2984)) and, when two complementaryDNA strands pair with a DNA duplex, a classical Holliday recombinationjoint or chi structure (Holliday, R. (1964) Genet. Res. 5: 282) canform, or a double-D loop (“Diagnostic Applications of Double-D LoopFormation” U.S. Ser. No. 07/755,462, filed Sep. 4, 1991). Once formed, aheteroduplex structure can be resolved by strand breakage and exchange,so that all or a portion of an invading DNA strand is spliced into arecipient DNA duplex, adding or replacing a segment of the recipient DNAduplex. Alternatively, a heteroduplex structure can result in geneconversion, wherein a sequence of an invading strand is transferred to arecipient DNA duplex by repair of mismatched bases using the invadingstrand as a template (Genes, 3rd Ed. (1987) Lewin, B., John Wiley, NewYork, N.Y.; Lopez et al. (1987) Nucleic Acids Res. 15: 5643). Whether bythe mechanism of breakage and rejoining or by the mechanism(s) of geneconversion, formation of heteroduplex DNA at homologously paired jointscan serve to transfer genetic sequence information from one DNA moleculeto another.

The ability of homologous recombination (gene conversion and classicalstrand breakage/rejoining) to transfer genetic sequence informationbetween DNA molecules renders targeted homologous recombination apowerful method in genetic engineering and gene manipulation.

In homologous recombination, the incoming DNA interacts with andintegrates into a site in the genome that contains a substantiallyhomologous DNA sequence. In non-homologous (“random” or “illicit”)integration, the incoming DNA is not found at a homologous sequence inthe genome but integrates elsewhere, at one of a large number ofpotential locations. In general, studies with higher eukaryotic cellshave revealed that the frequency of homologous recombination is far lessthan the frequency of random integration. The ratio of these frequencieshas direct implications for “gene targeting” which depends onintegration via homologous recombination (i.e. recombination between theexogenous “targeting DNA” and the corresponding “target DNA” in thegenome). The present invention can use homologous recombination toinactivate a gene or insert and upregulate or activate a gene in cells,such as the cells described above. The DNA can comprise at least aportion of the gene(s) at the particular locus with introduction of analteration into at least one, optionally both copies, of the nativegene(s), so as to prevent expression of functional gene product. Thealteration can be an insertion, deletion, replacement, mutation orcombination thereof. When the alteration is introduced into only onecopy of the gene being inactivated, the cells having a single unmutatedcopy of the target gene are amplified and can be subjected to a secondtargeting step, where the alteration can be the same or different fromthe first alteration, usually different, and where a deletion, orreplacement is involved, can be overlapping at least a portion of thealteration originally introduced. In this second targeting step, atargeting vector with the same arms of homology, but containing adifferent mammalian selectable markers can be used. The resultingtransformants are screened for the absence of a functional targetantigen and the DNA of the cell can be further screened to ensure theabsence of a wild-type target gene. Alternatively, homozygosity as to aphenotype can be achieved by breeding hosts heterozygous for themutation.

A number of papers describe the use of homologous recombination inmammalian cells. Illustrative of these papers are Kucherlapati et al.(1984) Proc. Natl. Acad. Sci. USA 81:3153-3157; Kucherlapati et al.(1985) Mol. Cell. Bio. 5:714-720; Smithies et al. (1985) Nature317:230-234; Wake et al. (1985) Mol. Cell. Bio. 8:2080-2089; Ayares etal. (1985) Genetics 111:375-388; Ayares et al. (1986) Mol. Cell. Bio.7:1656-1662; Song et al. (1987) Proc. Natl. Acad. Sci. USA 84:6820-6824;Thomas et al. (1986) Cell 44:419-428; Thomas and Capecchi, (1987) Cell51: 503-512; Nandi et al. (1988) Proc. Natl. Acad. Sci. USA85:3845-3849; and Mansour et al. (1988) Nature 336:348-352; Evans andKaufman, (1981) Nature 294:146-154; Doetschman et al. (1987) Nature330:576-578; Thoma and Capecchi, (1987) Cell 51:503-512; Thompson et al.(1989) Cell 56:316-321.

Gene Knockdown/Knockout Via RNAi

An alternative technology for disrupting the expression of a gene is RNAinterference. Interfering RNA (iRNA or siRNA) was originally describedin the model organism C. elegans (Fire et al., Nature 391:806-811(1998); U.S. Pat. No. 6,506,559 to Fire et al.). U.S. Pat. No. 6,573,099and PCT Publication No. WO 99/49029 by Benitec Australia Ltd. claimisolated genetic constructs which are capable of delaying, repressing orotherwise reducing the expression of a target gene in an animal cellwhich is transfected with the genetic construct, wherein the geneticconstruct contains at least two copies of a structural gene sequence.The structural gene sequence is described as a nucleotide sequence whichis substantially identical to at least a region of the target gene, andwherein at least two copies of the structural gene sequence are placedoperably under the control of a single promoter sequence such that atleast one copy of the structural gene sequence is placed operably in thesense orientation under the control of the promoter sequence. In thefield of xenotransplantation, DNA constructs driving expression ofsiRNA's was used to knock down the expression of porcine endogenousretrovirous (PERV) in transgenic pigs, see for example Ramsoondar etal., Xenotransplantation. 2009 May-June; 16(3):164-80; Dieckhoff et al.,Xenotransplantation. 2008 February; 15(1):36-45). siRNA technology hasalso been used to knock down alpha1,3 galactosyltransferase in porcinecells in vitro (Zhu et al., Transplantation. 2005 Feb. 15;79(3):289-96).

Random Insertion

In one embodiment, the DNA encoding the transgene sequences can berandomly inserted into the chromosome of a cell. The random integrationcan result from any method of introducing DNA into the cell known to oneof skill in the art. This may include, but is not limited to,electroporation, sonoporation, use of a gene gun, lipotransfection,calcium phosphate transfection, use of dendrimers, microinjection, theuse of viral vectors including adenoviral, AAV, and retroviral vectors,and group II ribozymes. In one embodiment, the DNA encoding the can bedesigned to include a reporter gene so that the presence of thetransgene or its expression product can be detected via the activationof the reporter gene. Any reporter gene known in the art can be used,such as those disclosed above. By selecting in cell culture those cellsin which the reporter gene has been activated, cells can be selectedthat contain the transgene. In other embodiments, the DNA encoding thetransgene can be introduced into a cell via electroporation. In otherembodiments, the DNA can be introduced into a cell via lipofection,infection, or transformation. In one embodiment, the electroporationand/or lipofection can be used to transfect fibroblast cells. In aparticular embodiment, the transfected fibroblast cells can be used asnuclear donors for nuclear transfer to generate transgenic animals asknown in the art and described below.

Cells that have been stained for the presence of a reporter gene canthen be sorted by FACS to enrich the cell population such that we have ahigher percentage of cells that contain the DNA encoding the transgeneof interest. In other embodiments, the FACS-sorted cells can then becultured for a periods of time, such as 12, 24, 36, 48, 72, 96 or morehours or for such a time period to allow the DNA to integrate to yield astable transfected cell population.

Vectors for Producing Transgenic Animals

Nucleic acid targeting vector constructs can be designed to accomplishhomologous recombination in cells. In one embodiment, a targeting vectoris designed using a “poly(A) trap”. Unlike a promoter trap, a poly(A)trap vector captures a broader spectrum of genes including those notexpressed in the target cell (i.e fibroblasts or ES cells). A polyA trapvector includes a constitutive promoter that drives expression of aselectable marker gene lacking a polyA signal. Replacing the polyAsignal is a splice donor site designed to splice into downstream exons.In this strategy, the mRNA of the selectable marker gene can bestabilized upon trapping of a polyA signal of an endogenous generegardless of its expression status in the target cells. In oneembodiment, a targeting vector is constructed including a selectablemarker that is deficient of signals for polyadenylation.

These targeting vectors can be introduced into mammalian cells by anysuitable method including, but not limited, to transfection,transformation, virus-mediated transduction, or infection with a viralvector. In one embodiment, the targeting vectors can contain a 3′recombination arm and a 5′ recombination arm (i.e. flanking sequence)that is homologous to the genomic sequence of interest. The 3′ and 5′recombination arms can be designed such that they flank the 3′ and 5′ends of at least one functional region of the genomic sequence. Thetargeting of a functional region can render it inactive, which resultsin the inability of the cell to produce functional protein. In anotherembodiment, the homologous DNA sequence can include one or more intronand/or exon sequences. In addition to the nucleic acid sequences, theexpression vector can contain selectable marker sequences, such as, forexample, enhanced Green Fluorescent Protein (eGFP) gene sequences,initiation and/or enhancer sequences, poly A-tail sequences, and/ornucleic acid sequences that provide for the expression of the constructin prokaryotic and/or eukaryotic host cells. The selectable marker canbe located between the 5′ and 3′ recombination arm sequence.

Modification of a targeted locus of a cell can be produced byintroducing DNA into the cells, where the DNA has homology to the targetlocus and includes a marker gene, allowing for selection of cellscomprising the integrated construct. The homologous DNA in the targetvector will recombine with the chromosomal DNA at the target locus. Themarker gene can be flanked on both sides by homologous DNA sequences, a3′ recombination arm and a 5′ recombination arm. Methods for theconstruction of targeting vectors have been described in the art, see,for example, Dai et al., Nature Biotechnology 20: 251-255, 2002; WO00/51424.

A variety of enzymes can catalyze the insertion of foreign DNA into ahost genome. Viral integrases, transposases and site-specificrecombinases mediate the integration of virus genomes, transposons orbacteriophages into host genomes. An extensive collection of enzymeswith these properties can be derived from a wide variety of sources.Retroviruses combine several useful features, including the relativesimplicity of their genomes, ease of use and their ability to integrateinto the host cell genome, permitting long-term transgene expression inthe transduced cells or their progeny. They have, therefore, been usedin a large number of gene-therapy protocols. Vectors based on Lentivirusvectors, have been attractive candidates for both gene therapy andtransgenic applications as have adeno-associated virus, which is a smallDNA virus (parvovirus) that is co-replicated in mammalian cells togetherwith helper viruses such as adenovirus, herpes simplex virus or humancytomegalovirus. The viral genome essentially consists of only two ORFs(rep, a non-structural protein, and cap, a structural protein) fromwhich (at least) seven different polypeptides are derived by alternativesplicing and alternative promoter usage. In the presence of ahelper-virus, the rep proteins mediate replication of the AAV genome.Integration, and thus a latent virus infection, occurs in the absence ofhelper virus. Transposons are also of interest. These are segments ofmobile DNA that can be found in a variety of organisms. Although activetransposons are found in many prokaryotic systems and insects, nofunctional natural transposons exist in vertebrates. The Drosophila Pelement transposon has been used for many years as a genome engineeringtool. The sleeping beauty transposon was established from non-functionaltransposon copies found in salmonid fish and is significantly moreactive in mammalian cells than prokaryotic or insect transposons.Site-specific recombinases are enzymes that catalyze DNA strand exchangebetween DNA segments that possess only a limited degree of sequencehomology. They bind to recognition sequences that are between 30 and 200nucleotides in length, cleave the DNA backbone, exchange the two DNAdouble helices involved and relegate the DNA. In some site-specificrecombination systems, a single polypeptide is sufficient to perform allof these reactions, whereas other recombinases require a varying numberof accessory proteins to fulfill these tasks. Site-specific recombinasescan be clustered into two protein families with distinct biochemicalproperties, namely tyrosine recombinases (in which the DNA is covalentlyattached to a tyrosine residue) and serine recombinases (where covalentattachment occurs at a serine residue). The most popular enzymes usedfor genome modification approaches are Cre (a tyrosine recombinasederived from E. coli bacteriophage P1) and fC31 integrase (a serinerecombinase derived from the Streptomyces phage fC31). Several otherbacteriophage derived site-specific recombinases (including Flp, lambdaintegrase, bacteriophage HK022 recombinase, bacteriophage R4 integraseand phage TP901-1 integrase) have been used successfully to mediatestable gene insertions into mammalian genomes. Recently, a site-specificrecombinase has been purified from the Streptomyces bacteriophage. ThefC31 recombinase is a member of the resolvase family and mediates phageintegration. In this process the bacteriophage attP site recombines withthe corresponding attB site in the bacterial genome. The crossovergenerates two sites, attL and attR, which are no longer a target forrecombinase action, in the absence of accessory proteins. The reactionalso takes place in mammalian cells and can therefore be used to mediatesite-specific integration of therapeutic genes. The site-specificity oftyrosine-recombinases has been difficult to modify by direct proteinengineering because the catalytic domain and the DNA recognition domainare closely interwoven. Therefore, changes in specificity are oftenaccompanied by a loss in activity. Serine recombinases might be moreamenable to engineering and a hyperactive derivative of Tn3 resolvasehas been modified by exchange of the natural DBD for a zinc-fingerdomain of the human zinc-finger transcription factor Zif268. The DNAsite-specificity of the resulting chimeric protein, termed Z-resolvase,had been switched to that of Zif268. Zinc-finger proteins can bemodified by in vitro protein evolution to recognize any DNA sequence,therefore, this approach could enable development of chimericrecombinases that can integrate therapeutic genes into precise genomiclocations. Methods for enhancing or mediating recombination include thecombination of site-specific recombination and homologous recombination,AAV-vector mediated, and zinc-finger nuclease mediated recombination(ref: Geurts et. al., Science, 325: 433, 2009)

The term “vector,” as used herein, refers to a nucleic acid molecule(preferably DNA) that provides a useful biological or biochemicalproperty to an inserted nucleic acid. “Expression vectors” according tothe invention include vectors that are capable of enhancing theexpression of one or more molecules that have been inserted or clonedinto the vector, upon transformation of the vector into a cell. Examplesof such expression vectors include, phages, autonomously replicatingsequences (ARS), centromeres, and other sequences which are able toreplicate or be replicated in vitro or in a cell, or to convey a desirednucleic acid segment to a desired location within a cell of an animal.Expression vectors useful in the present invention include chromosomal-,episomal- and virus-derived vectors, e.g., vectors derived frombacterial plasmids or bacteriophages, and vectors derived fromcombinations thereof, such as cosmids and phagemids or virus-basedvectors such as adenovirus, AAV, lentiviruses. A vector can have one ormore restriction endonuclease recognition sites at which the sequencescan be cut in a determinable fashion without loss of an essentialbiological function of the vector, and into which a nucleic acidfragment can be spliced in order to bring about its replication andcloning. Vectors can further provide primer sites, e.g., for PCR,transcriptional and/or translational initiation and/or regulation sites,recombinational signals, replicons, selectable markers, etc. Clearly,methods of inserting a desired nucleic acid fragment which do notrequire the use of homologous recombination, transpositions orrestriction enzymes (such as, but not limited to, UDG cloning of PCRfragments (U.S. Pat. No. 5,334,575), TA Cloning® brand PCR cloning(Invitrogen Corp., Carlsbad, Calif.)) can also be applied to clone anucleic acid into a vector to be used according to the presentinvention.

Cells homozygous at a targeted locus can be produced by introducing DNAinto the cells, where the DNA has homology to the target locus andincludes a marker gene, allowing for selection of cells comprising theintegrated construct. The homologous DNA in the target vector willrecombine with the chromosomal DNA at the target locus. The marker genecan be flanked on both sides by homologous DNA sequences, a 3′recombination arm and a 5′ recombination arm. Methods for theconstruction of targeting vectors have been described in the art, see,for example, Dai et al. (2002) Nature Biotechnology 20: 251-255; WO00/51424, FIG. 6; and Gene Targeting: A Practical Approach. Joyner, A.Oxford University Press, USA; 2^(nd) ed. Feb. 15, 2000.

Various constructs can be prepared for homologous recombination at atarget locus. Usually, the construct can include at least 25 bp, 50 bp,100 bp, 500 bp, 1 kbp, 2 kbp, 4 kbp, 5 kbp, 10 kbp, 15 kbp, 20 kbp, or50 kbp of sequence homologous with the target locus.

Various considerations can be involved in determining the extent ofhomology of target DNA sequences, such as, for example, the size of thetarget locus, availability of sequences, relative efficiency of doublecross-over events at the target locus and the similarity of the targetsequence with other sequences. The targeting DNA can include a sequencein which DNA substantially isogenic flanks the desired sequencemodifications with a corresponding target sequence in the genome to bemodified. The substantially isogenic sequence can be at least about 95%,97-98%, 99.0-99.5%, 99.6-99.9%, or 100% identical to the correspondingtarget sequence (except for the desired sequence modifications). Thetargeting DNA and the target DNA preferably can share stretches of DNAat least about 75, 150 or 500 base pairs that are 100% identical.Accordingly, targeting DNA can be derived from cells closely related tothe cell line being targeted; or the targeting DNA can be derived fromcells of the same cell line or animal as the cells being targeted.

Suitable selectable marker genes include, but are not limited to: genesconferring the ability to grow on certain media substrates, such as thetk gene (thymidine kinase) or the hprt gene (hypoxanthinephosphoribosyltransferase) which confer the ability to grow on HATmedium (hypoxanthine, aminopterin and thymidine); the bacterial gpt gene(guanine/xanthine phosphoribosyltransferase) which allows growth on MAXmedium (mycophenolic acid, adenine, and xanthine). See Song et al.(1987) Proc. Nat'l Acad. Sci. U.S.A. 84:6820-6824. See also Sambrook etal. (1989) Molecular Cloning—A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., see chapter 16. Other examples ofselectable markers include: genes conferring resistance to compoundssuch as antibiotics, genes conferring the ability to grow on selectedsubstrates, genes encoding proteins that produce detectable signals suchas luminescence, such as green fluorescent protein, enhanced greenfluorescent protein (eGFP). A wide variety of such markers are known andavailable, including, for example, antibiotic resistance genes such asthe neomycin resistance gene (neo) (Southern, P., and P. Berg, (1982) J.Mol. Appl. Genet. 1:327-341); and the hygromycin resistance gene (hyg)(Nucleic Acids Research 11:6895-6911 (1983), and Te Riele et al. (1990)Nature 348:649-651). Additional reporter genes useful in the methods ofthe present invention include acetohydroxyacid synthase (AHAS), alkalinephosphatase (AP), beta galactosidase (LacZ), beta glucuronidase (GUS),chloramphenicol acetyltransferase (CAT), green fluorescent protein(GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP),cyan fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase(Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivativesthereof. Multiple selectable markers are available that conferresistance to ampicillin, bleomycin, chloramphenicol, gentamycin,hygromycin, kanamycin, lincomycin, blasticidin, zeocin, methotrexate,phosphinothricin, puromycin, and tetracycline. Methods to determinesuppression of a reporter gene are well known in the art, and include,but are not limited to, fluorometric methods (e.g. fluorescencespectroscopy, Fluorescence Activated Cell Sorting (FACS), fluorescencemicroscopy), antibiotic resistance determination.

Combinations of selectable markers can also be used. To use acombination of markers, the HSV-tk gene can be cloned such that it isoutside of the targeting DNA (another selectable marker could be placedon the opposite flank, if desired). After introducing the DNA constructinto the cells to be targeted, the cells can be selected on theappropriate antibiotics. Selectable markers can also be used fornegative selection. Negative selection markets generally kill the cellsin which they are expressed either because the expression is per setoxic or produces a catalyst that leads to toxic metabolite, such asHerpes simplex virus Type I thymidine kinase (HSV-tk) or diphtheriatoxin A. Generally, the negative selection marker is incorporated intothe targeting vector so that it is lost following a preciserecombination event. Similarly, conventional selectable markers such asGFP can be used for negative selection using, for example, FACS sorting.

Deletions can be at least about 50 bp, more usually at least about 100bp, and generally not more than about 20 kbp, where the deletion cannormally include at least a portion of the coding region including aportion of or one or more exons, a portion of or one or more introns,and can or can not include a portion of the flanking non-coding regions,particularly the 5-non-coding region (transcriptional regulatoryregion). Thus, the homologous region can extend beyond the coding regioninto the 5′-non-coding region or alternatively into the 3-non-codingregion. Insertions can generally not exceed 10 kbp, usually not exceed 5kbp, generally being at least 50 bp, more usually at least 200 bp.

The region(s) of homology can include mutations, where mutations canfurther inactivate the target gene, in providing for a frame shift, orchanging a key amino acid, or the mutation can correct a dysfunctionalallele, etc. Usually, the mutation can be a subtle change, not exceedingabout 5% of the homologous flanking sequences or even a singlenucleotide change such as a point mutation in an active site of an exon.Where mutation of a gene is desired, the marker gene can be insertedinto an intron, so as to be excised from the target gene upontranscription.

Various considerations can be involved in determining the extent ofhomology of target DNA sequences, such as, for example, the size of thetarget locus, availability of sequences, relative efficiency of doublecross-over events at the target locus and the similarity of the targetsequence with other sequences. The targeting DNA can include a sequencein which DNA substantially isogenic flanks the desired sequencemodifications with a corresponding target sequence in the genome to bemodified. The substantially isogenic sequence can be at least about 95%,or at least about 97% or at least about 98% or at least about 99% orbetween 95 and 100%, 97-98%, 99.0-99.5%, 99.6-99.9%, or 100% identicalto the corresponding target sequence (except for the desired sequencemodifications). In a particular embodiment, the targeting DNA and thetarget DNA can share stretches of DNA at least about 75, 150 or 500 basepairs that are 100% identical. Accordingly, targeting DNA can be derivedfrom cells closely related to the cell line being targeted; or thetargeting DNA can be derived from cells of the same cell line or animalas the cells being targeted.

The construct can be prepared in accordance with methods known in theart, various fragments can be brought together, introduced intoappropriate vectors, cloned, analyzed and then manipulated further untilthe desired construct has been achieved. Various modifications can bemade to the sequence, to allow for restriction analysis, excision,identification of probes, etc. Silent mutations can be introduced, asdesired. At various stages, restriction analysis, sequencing,amplification with the polymerase chain reaction, primer repair, invitro mutagenesis, etc. can be employed.

The construct can be prepared using a bacterial vector, including aprokaryotic replication system, e.g. an origin recognizable by E. coli,at each stage the construct can be cloned and analyzed. A marker, thesame as or different from the marker to be used for insertion, can beemployed, which can be removed prior to introduction into the targetcell. Once the vector containing the construct has been completed, itcan be further manipulated, such as by deletion of the bacterialsequences, linearization, introducing a short deletion in the homologoussequence. After final manipulation, the construct can be introduced intothe cell.

Techniques which can be used to allow the DNA or RNA construct entryinto the host cell include calcium phosphate/DNA coprecipitation,microinjection of DNA into the nucleus, electroporation, bacterialprotoplast fusion with intact cells, transfection, lipofection,infection, particle bombardment, sperm mediated gene transfer, or anyother technique known by one skilled in the art. The DNA or RNA can besingle or double stranded, linear or circular, relaxed or supercoiledDNA. For various techniques for transfecting mammalian cells, see, forexample, Keown et al., Methods in Enzymology Vol. 185, pp. 527-537(1990).

The following vectors are provided by way of example. Bacterial: pBs,pQE-9 (Qiagen), phagescript, PsiXl74, pBluescript SK, pBsKS, pNH8a,pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3,pDR54O, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSv2cat, pOG44, pXT1, pSG(Stratagene) pSVK3, pBPv, pMSG, pSVL (Pharmiacia). Also, any otherplasmids and vectors can be used as long as they are replicable andviable in the host. Vectors known in the art and those commerciallyavailable (and variants or derivatives thereof) can in accordance withthe invention be engineered to include one or more recombination sitesfor use in the methods of the invention. Such vectors can be obtainedfrom, for example, Vector Laboratories Inc., Invitrogen, Promega,Novagen, NEB, Clontech, Boehringer Mannheim, Pharmacia, EpiCenter,OriGenes Technologies Inc., Stratagene, PerkinElmer, Pharmingen, andResearch Genetics. Other vectors of interest include eukaryoticexpression vectors such as pFastBac, pFastBacHT, pFastBacDUAL, pSFV, andpTet-Splice (Invitrogen), pEUK-C1, pPUR, pMAM, pMAMneo, pBI101, pBI121,pDR2, pCMVEBNA, and pYACneo (Clontech), pSVK3, pSVL, pMSG, pCH110, andpKK232-8 (Pharmacia, Inc.), p3'SS, pXT1, pSG5, pPbac, pMbac, pMC1neo,and pOG44 (Stratagene, Inc.), and pYES2, pAC360, pBlueBacHis A, B, andC, pVL1392, pBlueBacIII, pCDM8, pcDNA1, pZeoSV, pcDNA3 pREP4, pCEP4, andpEBVHis (Invitrogen, Corp.) and variants or derivatives thereof.

Other vectors include pUC18, pUC19, pBlueScript, pSPORT, cosmids,phagemids, YAC's (yeast artificial chromosomes), BAC's (bacterialartificial chromosomes), P1 (Escherichia coli phage), pQE70, pQE60, pQE9(quagan), pBS vectors, PhageScript vectors, BlueScript vectors, pNH8A,pNH16A, pNH18A, pNH46A (Stratagene), pcDNA3 (Invitrogen), pGEX, pTrsfus,pTrc99A, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia),pSPORT1, pSPORT2, pCMVSPORT2.0 and pSY-SPORT1 (Invitrogen) and variantsor derivatives thereof. Viral vectors can also be used, such aslentiviral vectors (see, for example, WO 03/059923; Tiscornia et al.PNAS 100:1844-1848 (2003)).

Additional vectors of interest include pTrxFus, pThioHis, pLEX, pTrcHis,pTrcHis2, pRSET, pBlueBacHis2, pcDNA3.1/His, pcDNA3.1(−)/Myc-His,pSecTag, pEBVHis, pPIC9K, pPIC3.5K, pAO81S, pPICZ, pPICZA, pPICZB,pPICZC, pGAPZA, pGAPZB, pGAPZC, pBlueBac4.5, pBlueBacHis2, pMelBac,pSinRep5, pSinHis, pIND, pIND(SP1), pVgRXR, pcDNA2.1, pYES2, pZErO1.1,pZErO-2.1, pCR-Blunt, pSE280, pSE380, pSE420, pVL1392, pVL1393, pCDM8,pcDNA1.1, pcDNA1.1/Amp, pcDNA3.1, pcDNA3.1/Zeo, pSe, SV2, pRc/CMV2,pRc/RSV, pREP4, pREP7, pREP8, pREP9, pREP 10, pCEP4, pEBVHis, pCR3.1,pCR2.1, pCR3.1-Uni, and pCRBac from Invitrogen; λ ExCell, λ gt11,pTrc99A, pKK223-3, pGEX-1λT, pGEX-2T, pGEX-2TK, pGEX-4T-1, pGEX-4T-2,pGEX-4T-3, pGEX-3×, pGEX-5X-1, pGEX-5X-2, pGEX-5X-3, pEZZ18, pRIT2T,pMC1871, pSVK3, pSVL, pMSG, pCH110, pKK232-8, pSL1180, pNEO, and pUC4Kfrom Pharmacia; pSCREEN-1b(+), pT7Blue(R), pT7Blue-2, pCITE-4-abc(+),pOCUS-2, pTAg, pET-32L1C, pET-30LIC, pBAC-2 cp LIC, pBACgus-2 cp LIC,pT7Blue-2 LIC, pT7Blue-2, λ SCREEN-1, X BlueSTAR, pET-3abcd, pET-7abc,pET9abcd, pET11abcd, pET12abc, pET-14b, pET-15b, pET-16b,pET-17b-pET-17xb, pET-19b, pET-20b(+), pET-21abcd(+), pET-22b(+),pET-23abcd(+), pET-24abcd(+), pET-25b(+), pET-26b(+), pET-27b(+),pET-28abc(+), pET-29abc(+), pET-30abc(+), pET-31b(+), pET-32abc(+),pET-33b(+), pBAC-1, pBACgus-1, pBAC4x-1, pBACgus4x-1, pBAC-3 cp,pBACgus-2 cp, pBACsurf-1, plg, Signal plg, pYX, Selecta Vecta-Neo,Selecta Vecta-Hyg, and Selecta Vecta-Gpt from Novagen; pLexA, pB42AD,pGBT9, pAS2-1, pGAD424, pACT2, pGAD GL, pGAD GH, pGAD10, pGilda, pEZM3,pEGFP, pEGFP-1, pEGFP-N, pEGFP-C, pEBFP, pGFPuv, pGFP, p6xHis-GFP,pSEAP2-Basic, pSEAP2-Contral, pSEAP2-Promoter, pSEAP2-Enhancer,pβgal-Basic, pβgal-Control, pβgal-Promoter, pβgal-Enhancer, pCMV,pTet-Off, pTet-On, pTK-Hyg, pRetro-Off, pRetro-On, pIRES1neo, pIRES1hyg,pLXSN, pLNCX, pLAPSN, pMAMneo, pMAMneo-CAT, pMAMneo-LUC, pPUR, pSV2neo,pYEX4T-1/2/3, pYEX-S1, pBacPAK-His, pBacPAK8/9, pAcUW31, BacPAK6,pTriplEx, λgt10, λgt11, pWE15, and XTriplEx from Clontech; Lambda ZAPII, pBK-CMV, pBK-RSV, pBluescript II KS +/−, pBluescript II SK +/−,pAD-GAL4, pBD-GAL4 Cam, pSurfscript, Lambda FIX II, Lambda DASH, LambdaEMBL3, Lambda EMBL4, SuperCos, pCR-Script Amp, pCR-Script Cam,pCR-Script Direct, pBS +/−, pBC KS +/−, pBC SK +/−, Phagescript,pCAL-n-EK, pCAL-n, pCAL-c, pCAL-kc, pET-3abcd, pET-11abcd, pSPUTK,pESP-1, pCMVLacI, pOPRSVI/MCS, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac,pMC1neo, pMC1neo Poly A, pOG44, pOG45, pFRTβGAL, pNEOβGAL, pRS403,pRS404, pRS405, pRS406, pRS413, pRS414, pRS415, and pRS416 fromStratagene.

Additional vectors include, for example, pPC86, pDBLeu, pDBTrp, pPC97,p2.5, pGAD1-3, pGAD10, pACt, pACT2, pGADGL, pGADGH, pAS2-1, pGAD424,pGBT8, pGBT9, pGAD-GAL4, pLexA, pBD-GAL4, pHISi, pHISi-1, placZi,pB42AD, pDG202, pJK202, pJG4-5, pNLexA, pYESTrp and variants orderivatives thereof.

Promoters

Vector constructs used to produce the animals of the invention caninclude regulatory sequences, including, for example, a promoter,operably linked to the sequence. Large numbers of suitable vectors andpromoters are known to those of skill in the art, and are commerciallyavailable.

In specific embodiments, the present invention provides animals, organs,tissues and cells that express a transgene, and in particular animmunomodulator or anticoagulant transgene, in endothelium. To targetexpression to a particular tissue, the animal is developed using avector that includes a promoter specific for endothelial geneexpression.

In one embodiment, the nucleic acid construct contains a regulatorysequence operably linked to the transgene sequence to be expressed. Inone embodiment, the regulatory sequence can be a promoter sequence. Inone embodiment, the promoter can be a regulatable promoter. In suchsystems, drugs, for example, can be used to regulate whether the peptideis expressed in the animal, tissue or organ. For example, expression canbe prevented while the organ or tissue is part of the pig, butexpression induced once the pig has been transplanted to the human for aperiod of time to overcome the cellular immune response. In addition,the level of expression can be controlled by a regulatable promotersystem to ensure that immunosuppression of the recipient's immune systemdoes not occur. The regulatable promoter system can be selected from,but not limited to, the following gene systems: a metallothioneinpromoter, inducible by metals such as copper (see Lichtlen andSchaffner, Swiss Med. Wkly., 2001, 131 (45-46):647-52); atetracycline-regulated system (see Imhof et al., J Gene Med., 2000,2(2):107-16); an ecdysone-regulated system (see Saez et al., Proc NatlAcad Sci USA., 2000, 97(26):14512-7); a cytochrome P450 induciblepromoter, such as the CYP1A1 promoter (see Fujii-Kuriyama et al., FASEBJ., 1992, 6(2):706-10); a mifepristone inducible system (see Sirin andPark, Gene., 2003, 323:67-77); a coumarin-activated system (see Zhao etal., Hum Gene Ther., 2003, 14(17): 1619-29); a macrolide induciblesystem (responsive to macrolide antibiotics such as rapamycin,erythromycin, clarithromycin, and roxithromycin) (see Weber et al., Nat.Biotechnol., 2002, 20(9):901-7; Wang et al., Mol Ther., 2003,7(6):790-800); an ethanol induced system (see Garoosi et al., J ExpBot., 2005, 56(416):163542; Roberts et al., Plant Physiol., 2005,138(3):1259-67); a streptogramin inducible system (see Fussenegger etal., Nat Biotechnol., 2000 18(11):1203-8) an electrophile induciblesystem (see Zhu and Fahl, Biochem Biophys Res Commun., 2001,289(1):212-9); and a nicotine inducible system (see Malphettes et al.,Nucleic Acids Res., 2005, 33(12):e107).

In particular embodiments, the promoter is a tissue specific promotersuch as those described herein. The tissue specific promoter can be usedin particular for the expression of an anticoagulant orimmunosuppressant. The tissue specific promoter is most preferably aendothelial-specific promoter. In one embodiment, theendothelial-specific promoter is the mouse Tie-2 promoter (see, forexample, Schlaeger et al., 1997 Proc Natl Acad Sci USA. April 1;94(7):3058-63). In another embodiment, the endothelial-specific promoteris the porcine ICAM-2 promoter (see, for example, Godwin et al., 2006.Xenotransplantation. November; 13(6):514-21). In other embodiments anenhancer element is used in the nucleic acid construct to facilitateincreased expression of the transgene in a tissue-specific manner.Enhancers are outside elements that drastically alter the efficiency ofgene transcription (Molecular Biology of the Gene, Fourth Edition, pp.708-710, Benjamin Cummings Publishing Company, Menlo Park, Calif.©1987). In certain embodiments, the animal expresses a transgene underthe control of a promoter in combination with an enhancer element. Inparticular embodiments, the animal includes an endothelial specificpromoter element, such as a porcine ICAM-2 or murine Tie-2 promoter, andfurther includes an enhancer element. In some embodiments, the promoteris used in combination with an enhancer element which is a non-coding orintronic region of DNA intrinsically associated or co-localized with thepromoter. In a specific embodiment, the enhancer element is Tie-2 usedin combination with the Tie-2 promoter. In another specific embodiment,the enhancer element is ICAM-2 used in combination with the ICAM-2promoter. In other embodiments, the promoter can be a ubiquitouspromoter. Ubiquitous promoters include, but are not limited to thefollowing: viral promoters like CMV, SV40. Suitable promoters alsoinclude beta-Actin promoter, gamma-actin promoter, GAPDH promoters, H₂K,ubiquitin and the rosa promoter.

Selection of Transgenic Cells

In some cases, the transgenic cells have genetic modifications that arethe result of targeted transgene insertion or integration (i.e. viahomologous recombination) into the cellular genome. In some cases, thetransgenic cells have genetic modification that are the result ofnon-targeted (random) integration into the cellular genome. The cellscan be grown in appropriately-selected medium to identify cellsproviding the appropriate integration. Those cells which show thedesired phenotype can then be further analyzed by restriction analysis,electrophoresis, Southern analysis, polymerase chain reaction, oranother technique known in the art. By identifying fragments which showthe appropriate insertion at the target gene site, (or, in non-targetedapplications, where random integration techniques have produced thedesired result,) cells can be identified in which homologousrecombination (or desired non-targeted integration events) has occurredto inactivate or otherwise modify the target gene.

The presence of the selectable marker gene establishes the integrationof the target construct into the host genome. Those cells which show thedesired phenotype can then be further analyzed by restriction analysis,electrophoresis, Southern analysis, polymerase chain reaction, etc toanalyze the DNA in order to establish whether homologous ornon-homologous recombination occurred. This can be determined byemploying probes for the insert and then sequencing the 5′ and 3′regions flanking the insert for the presence of the gene extendingbeyond the flanking regions of the construct or identifying the presenceof a deletion, when such deletion is introduced. Primers can also beused which are complementary to a sequence within the construct andcomplementary to a sequence outside the construct and at the targetlocus. In this way, one can only obtain DNA duplexes having both of theprimers present in the complementary chains if homologous recombinationhas occurred. For example, by demonstrating the presence of the primersequences or the expected size sequence, the occurrence of homologousrecombination is supported.

The polymerase chain reaction used for screening homologousrecombination events is described in Kim and Smithies, (1988) NucleicAcids Res. 16:8887-8903; and Joyner et al. (1989) Nature 338:153-156.

The cell lines obtained from the first round of targeting (or fromnon-targeted (random) integration into a desired location) are likely tobe heterozygous for the integrated allele. Homozygosity, in which bothalleles are modified, can be achieved in a number of ways. One approachis to grow up a number of cells in which one copy has been modified andthen to subject these cells to another round of targeting (ornon-targeted (random) integration) using a different selectable marker.Alternatively, homozygotes can be obtained by breeding animalsheterozygous for the modified allele. In some situations, it can bedesirable to have two different modified alleles. This can be achievedby successive rounds of gene targeting (or random integration) or bybreeding heterozygotes, each of which carries one of the desiredmodified alleles. In certain embodiments, at least one element of theanimal is derived by selection of a spontaneously occurring mutation inan allele, in particular to develop a homozygous animal. In certainembodiments, a selection technique is used to obtain homologous knockoutcells from heterozygous cells by exposure to very high levels of aselection agent. Such a selection can be, for example, by use of anantibiotic such as geneticin (G418).

Cells that have been transfected or otherwise received an appropriatevector can then be selected or identified via genotype or phenotypeanalysis. In one embodiment, cells are transfected, grown inappropriately-selected medium to identify cells containing theintegrated vector. The presence of the selectable marker gene indicatesthe presence of the transgene construct in the transfected cells. Thosecells which show the desired phenotype can then be further analyzed byrestriction analysis, electrophoresis, Southern analysis, polymerasechain reaction, etc to analyze the DNA in order to verify integration oftransgene(s) into the genome of the host cells. Primers can also be usedwhich are complementary to transgene sequence(s). The polymerase chainreaction used for screening homologous recombination and randomintegration events is known in the art, see, for example, Kim andSmithies, Nucleic Acids Res. 16:8887-8903, 1988; and Joyner et al.,Nature 338:153-156, 1989. The specific combination of a mutant polyomaenhancer and a thymidine kinase promoter to drive the neomycin gene hasbeen shown to be active in both embryonic stem cells and EC cells byThomas and Capecchi, supra, 1987; Nicholas and Berg (1983) inTeratocarcinoma Stem Cell, eds. Siver, Martin and Strikland (Cold SpringHarbor Lab., Cold Spring Harbor, N.Y. (pp. 469-497); and Linney andDonerly, Cell 35:693-699, 1983.

Cells that have undergone homologous recombination can be identified bya number of methods. In one embodiment, the selection method can detectthe absence of an immune response against the cell, for example by ahuman anti-gal antibody. In other embodiments, the selection method caninclude assessing the level of clotting in human blood when exposed to acell or tissue. Selection via antibiotic resistance has been used mostcommonly for screening. This method can detect the presence of theresistance gene on the targeting vector, but does not directly indicatewhether integration was a targeted recombination event or a randomintegration. Alternatively, the marker can be a fluorescent marker genesuch as GFP or RFP, or a gene that is detectable on the cell surface viacell sorting or FACs analysis. Certain technology, such as Poly A andpromoter trap technology, increase the probability of targeted events,but again, do not give direct evidence that the desired phenotype hasbeen achieved. In addition, negative forms of selection can be used toselect for targeted integration; in these cases, the gene for a factorlethal to the cells (e.g. Tk or diphtheria A toxin) is inserted in sucha way that only targeted events allow the cell to avoid death. Cellsselected by these methods can then be assayed for gene disruption,vector integration and, finally, gene depletion. In these cases, sincethe selection is based on detection of targeting vector integration andnot at the altered phenotype, only targeted knockouts, not pointmutations, gene rearrangements or truncations or other suchmodifications can be detected.

Characterization can be further accomplished by the followingtechniques, including, but not limited to: PCR analysis, Southern blotanalysis, Northern blot analysis, specific lectin binding assays, and/orsequencing analysis. Phenotypic characterization can also beaccomplished, including by binding of anti-mouse antibodies in variousassays including immunofluorescence, immunocytochemistry, ELISA assays,flow cytometry, western blotting, testing for transcription of RNA incells such as by RT-PCR.

In other embodiments, GTKO animals or cells contain additional geneticmodifications. Genetic modifications can include more than justhomologous targeting, but can also include random integrations ofexogenous genes, mutations, deletions and insertions of genes of anykind. The additional genetic modifications can be made by furthergenetically modifying cells obtained from the transgenic cells andanimals described herein or by breeding the animals described hereinwith animals that have been further genetically modified. Such animalscan be modified to eliminate the expression of at least one allele ofαGT gene, the CMP-Neu5Ac hydroxylase gene (see, for example, U.S. Pat.No. 7,368,284), the iGb3 synthase gene (see, for example, U.S. PatentPublication No. 2005/0155095), and/or the Forssman synthase gene (see,for example, U.S. Patent Publication No. 2006/0068479). In additionalembodiments, the animals described herein can also contain geneticmodifications to express fucosyltransferase, sialyltransferase and/orany member of the family of glucosyltransferases. To achieve theseadditional genetic modifications, in one embodiment, cells can bemodified to contain multiple genetic modifications. In otherembodiments, animals can be bred together to achieve multiple geneticmodifications. In one specific embodiment, animals, such as pigs,lacking expression of functional immunoglobulin, produced according tothe process, sequences and/or constructs described herein, can be bredwith animals, such as pigs, lacking expression of αGT (for example, asdescribed in WO 04/028243).

In another embodiment, the expression of additional genes responsiblefor xenograft rejection can be eliminated or reduced. Such genesinclude, but are not limited to the CMP-NEUAc Hydroxylase Gene, theisoGloboside 3 Synthase gene, and the Forssman synthase gene.

In addition, genes or cDNA encoding complement related proteins, whichare responsible for the suppression of complement mediated lysis canalso be expressed in the animals and tissues of the present invention.Such genes include, but are not limited to CD59, DAF (CD55), and CD46(see, for example, WO 99/53042; Chen et al. Xenotransplantation, Volume6 Issue 3 Page 194-August 1999, which describes pigs that expressCD59/DAF transgenes; Costa C et al, Xenotransplantation. 2002 January;9(1):45-57, which describes transgenic pigs that express human CD59 andH-transferase; Zhao L et al.; Diamond L E et al. Transplantation. 2001Jan. 15; 71(1):132-42, which describes a human CD46 transgenic pigs.)

Additional modifications can include expression of compounds, such asantibodies, which down-regulate the expression of a cell adhesionmolecule by the cells, such as described in WO 00/31126, entitled“Suppression of xenograft rejection by down regulation of a celladhesion molecules” and compounds in which co-stimulation by signal 2 isprevented, such as by administration to the organ recipient of a solubleform of CTLA-4 from the xenogeneic donor organism, for example asdescribed in WO 99/57266, entitled “Immunosuppression by blocking T cellco-stimulation signal 2 (B7/CD28 interaction)”.

Nuclear Transfer

Engineered transgenic animals such as ungulates or pigs described hereinmay be produced using any suitable techniques known in the art. Thesetechniques include, but are not limited to, microinjection (e.g., ofpronuclei), sperm-mediated gene transfer, electroporation of ova orzygotes, and/or nuclear transplantation.

In certain embodiments, sperm mediated gene transfer can be used toproduce the genetically modified ungulates described herein. The methodsand compositions described herein to insert transgenes can be used togenetically modify sperm cells via any technique described herein orknown in the art. The genetically modified sperm can then be used toimpregnate a female recipient via artificial insemination,intra-cytoplasmic sperm injection or any other known technique. In oneembodiment, the sperm and/or sperm head can be incubated with theexogenous nucleic acid for a sufficient time period. Sufficient timeperiods include, for example, about 30 seconds to about 5 minutes,typically about 45 seconds to about 3 minutes, more typically about 1minute to about 2 minutes.

The potential use of sperm cells as vectors for gene transfer was firstsuggested by Brackeff et al., Proc., Natl. Acad. Sci. USA 68:353-357(1971). This was followed by reports of the production of transgenicmice and pigs after in vitro fertilization of oocytes with sperm thathad been incubated by naked DNA (see, for example, Lavitrano et al.,Cell 57:717-723 (1989) and Gandolfi et al. Journal of Reproduction andFertility Abstract Series 4, 10 (1989)), although other laboratorieswere not able to repeat these experiments (see, for example, Brinster etal. Cell 59:239-241 (1989) and Gavora et al., Canadian Journal of AnimalScience 71:287-291 (1991)). Since then, successful sperm mediated genetransfer has been achieved in chicken (see, for example, Nakanishi andIritani, Mol. Reprod. Dev. 36:258-261 (1993)); mice (see, for example,Maione, Mol. Reprod. Dev. 59:406 (1998)); and pigs (see, for example,Lavitrano et al. Transplant. Proc. 29:3508-3509 (1997); Lavitrano etal., Proc. Natl. Acad. Sci. USA 99:14230-5 (2002); Lavitrano et al.,Mol. Reprod. Dev. 64-284-91 (2003)). Similar techniques are alsodescribed in U.S. Pat. No. 6,376,743; issued Apr. 23, 2002; U.S. PatentPublication Nos. 20010044937, published Nov. 22, 2001, and 20020108132,published Aug. 8, 2002).

In some embodiments, intracytoplasmic sperm injection can be used toproduce the genetically modified ungulates described herein. This can beaccomplished by coinserting an exogenous nucleic acid and a sperm intothe cytoplasm of an unfertilized oocyte to form a transgenic fertilizedoocyte, and allowing the transgenic fertilized oocyte to develop into atransgenic embryo and, if desired, into a live offspring. The sperm canbe a membrane-disrupted sperm head or a demembranated sperm head. Thecoinsertion step can include the substep of preincubating the sperm withthe exogenous nucleic acid for a sufficient time period, for example,about 30 seconds to about 5 minutes, typically about 45 seconds to about3 minutes, more typically about 1 minute to about 2 minutes. Thecoinsertion of the sperm and exogenous nucleic acid into the oocyte canbe via microinjection. The exogenous nucleic acid mixed with the spermcan contain more than one transgene, to produce an embryo that istransgenic for more than one transgene as described herein. Theintracytoplasmic sperm injection can be accomplished by any techniqueknown in the art, see, for example, U.S. Pat. No. 6,376,743.

Any additional technique known in the art may be used to introduce thetransgene into animals. Such techniques include, but are not limited topronuclear microinjection (see, for example, Hoppe, P. C. and Wagner, T.E., 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transferinto germ lines (see, for example, Van der Putten et al., 1985, Proc.Natl. Acad. Sci., USA 82:6148-6152); gene targeting in embryonic stemcells (see, for example, Thompson et al., 1989, Cell 56:313-321;Wheeler, M. B., 1994, WO 94/26884); electroporation of embryos (see, forexample, Lo, 1983, Mol Cell. Biol. 3:1803-1814); cell gun; transfection;transduction; retroviral infection; adenoviral infection;adenoviral-associated infection; liposome-mediated gene transfer; nakedDNA transfer; and sperm-mediated gene transfer (see, for example,Lavitrano et al., 1989, Cell 57:717-723); etc. For a review of suchtechniques, see, for example, Gordon, 1989, Transgenic Anithals, Intl.Rev. Cytol. 115:171-229. In particular embodiments, the expression ofCTLA4 and/or CTLA4-Ig fusion genes in ungulates can be accomplished viathese techniques.

In one embodiment, microinjection of the constructs encoding thetransgene can be used to produce the transgenic animals. In oneembodiment, the nucleic acid construct or vector can be microinjectioninto the pronuclei of a zygote. In one embodiment, the construct orvector can be injected into the male pronuclei of a zygote. In anotherembodiment, the construct or vector can be injected into the femalepronuclei of a zygote. In a further embodiment, the construct or vectorcan be injected via sperm-mediated gene transfer.

Microinjection of the transgene construct or vector can include thefollowing steps: superovulation of a donor female; surgical removal ofthe egg, fertilization of the egg; injection of the transgenetranscription unit into the pronuclei of the embryo; and introduction ofthe transgenic embryo into the reproductive tract of a pseudopregnanthost mother, usually of the same species. See for example U.S. Pat. No.4,873,191, Brinster, et al. 1985. PNAS 82:4438; Hogan, et al., in“Manipulating the Mouse Embryo: A Laboratory Manual”. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1986. Robertson, 1987, inRobertson, ed. “Teratocarcinomas and Embryonic Stem Cells a PracticalApproach” IRL Press, Evnsham. Oxford, England. Pedersen, et al., 1990.“Transgenic Techniques in Mice—A Video Guide”, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. Transgenic pigs are routinelyproduced by the microinjection of a transgene construct or vector intopig embryos. In one embodiment, the presence of the transgene can bedetected by isolating genomic DNA from tissue from the tail of eachpiglet and subjecting about 5 micrograms of this genomic DNA to nucleicacid hybridization analysis with a transgene specific probe. In aparticular embodiment, transgenic animals can be produced according toany method known to one skilled in the art, for example, as disclosed inBleck et al., J. Anim. Sci., 76:3072 [1998]; also described in U.S. Pat.Nos. 6,872,868; 6,066,725; 5,523,226; 5,453,457; 4,873,191; 4,736,866;and/or PCT Publication No. WO/9907829.

In one embodiment, the pronuclear microinjection method can includelinking at least approximately 50, 100, 200, 300, 400 or 500 copies ofthe transgene-containing construct or vector of the present invention toa promoter of choice, for example, as disclosed herein, and then theforeign DNA can be injected through a fine glass needle into fertilizedeggs. In one embodiment, the DNA can be injected into the malepronucleus of the zygote. Pig zygotes are opaque and visualization ofnuclear structures can be difficult. In one embodiment, the pronuclei ornuclei of pig zygotes can be visualized after centrifugation, forexample, at 15,000 g for 3 mm. The injection of the pronucleus can becarried out under magnification and use of standard microinjectionapparatus. The zygote can be held by a blunt holding pipette and thezona pellucida, plasma membrane and pronuclear envelope can bepenetrated by an injection pipette. The blunt holding pipette can have asmall diameter, for example, approximately 50 um. The injection pipettecan have a smaller diameter than the holding pipette, for example,approximately 15 um. DNA integration occurs during replication as arepair function of the host DNA. These eggs, containing the foreign DNA,can then be implanted into surrogate mothers for gestation of the embryoaccording to any technique known to one skilled in the art.

In some embodiments, pronuclear microinjection can be performed on thezygote 12 hours post fertilization. Uptake of such genes can be delayedfor several cell cycles. The consequence of this is that depending onthe cell cycle of uptake, only some cell lineages may carry thetransgene, resulting in mosaic offspring. If desired, mosaic animals canbe bred to form true germline transgenic animals.

In other embodiments, ungulate cells such as porcine cells containingtransgenes can be used as donor cells to provide the nucleus for nucleartransfer into enucleated oocytes to produce cloned, transgenic animals.In one embodiment, the ungulate cell need not express the transgeneprotein in order to be useful as a donor cell for nuclear transfer. Inone embodiment, the porcine cell can be engineered to express atransgene from a nucleic acid construct or vector that contains apromoter. Alternatively, the porcine cells can be engineered to expresstransgene under control of an endogenous promoter through homologousrecombination. In one embodiment, the transgene nucleic acid sequencecan be inserted into the genome under the control of a tissue specificpromoter, tissue specific enhancer or both. In another embodiment, thetransgene nucleic acid sequence can be inserted into the genome underthe control of a ubiquitous promoter. In certain embodiments, targetingvectors are provided, which are designed to allow targeted homologousrecombination in somatic cells. These targeting vectors can betransformed into mammalian cells to target the endogenous genes ofinterest via homologous recombination. In one embodiment, the targetingconstruct inserts both the transgene nucleotide sequence and aselectable maker gene into the endogenous gene so as to be in readingframe with the upstream sequence and produce an active fusion protein.Cells can be transformed with the constructs using the methods of theinvention and are selected by means of the selectable marker and thenscreened for the presence of recombinants.

The present invention provides a method for cloning an ungulate such asa pig containing certain transgenes via somatic cell nuclear transfer.In general, the pig can be produced by a nuclear transfer processcomprising the following steps: obtaining desired differentiated pigcells to be used as a source of donor nuclei; obtaining oocytes from apig; enucleating said oocytes; transferring the desired differentiatedcell or cell nucleus into the enucleated oocyte, e.g., by fusion orinjection, to form nuclear transfer (NT) units; activating the resultantNT unit; and transferring said cultured NT unit to a host pig such thatthe NT unit develops into a fetus.

Nuclear transfer techniques or nuclear transplantation techniques areknown in the art (see, for example, Dai et al. Nature Biotechnology20:251-255; Polejaeva et al Nature 407:86-90 (2000); Campbell, et al.,Theriogenology 68 Suppl 1:S214-3 1 (2007); Vajta, et al., Reprod FertilDev 19(2): 403-23 (2007); Campbell et al. (1995) Theriogenology, 43:181;Collas et al. (1994) Mol. Report Dev., 38:264-267; Keefer et al. (1994)Biol. Reprod., 50:935-939; Sims et al. (1993) Proc. Natl. Acad. Sci.,USA, 90:6143-6147; WO 94/26884; WO 94/24274, and WO 90/03432, U.S. Pat.Nos. 4,944,384, 5,057,420, WO 97/07669, WO 97/07668, WO 98/30683, WO00/22098, WO 004217, WO 00/51424, WO 03/055302, WO 03/005810, U.S. Pat.Nos. 6,147,276, 6,215,041, 6,235,969, 6,252,133, 6,258,998, 5,945,577,6,525,243, 6,548,741, and Phelps et al. (Science 299:411-414 (2003)).

A donor cell nucleus, which has been modified to contain a transgene ofthe present invention, is transferred to a recipient porcine oocyte. Theuse of this method is not restricted to a particular donor cell type.The donor cell can be as described in Wilmut et al. (1997) Nature385:810; Campbell et al. (1996) Nature 380:64-66; or Cibelli et al.(1998) Science 280:1256-1258. All cells of normal karyotype, includingembryonic, fetal and adult somatic cells which can be used successfullyin nuclear transfer can in principle be employed. Fetal fibroblasts area particularly useful class of donor cells. Generally suitable methodsof nuclear transfer are described in Campbell et al. (1995)Theriogenology 43:181, Collas et al. (1994) Mol. Reprod. Dev.38:264-267, Keefer et al. (1994) Biol. Reprod. 50:935-939, Sims et al.(1993) Proc. Nat'l. Acad. Sci. USA 90:6143-6147, WO-A-9426884,WO-A-9424274, WO-A-9807841, WO-A-9003432, U.S. Pat. Nos. 4,994,384 and5,057,420, Campbell et al., (2007) Theriogenology 68 Suppl 1, S214-231,Vatja et al., (2007) Reprod Fertil Dev 19, 403-423). Differentiated orat least partially differentiated donor cells can also be used. Donorcells can also be, but do not have to be, in culture and can bequiescent. Nuclear donor cells which are quiescent are cells which canbe induced to enter quiescence or exist in a quiescent state in vivo.Prior art methods have also used embryonic cell types in cloningprocedures (see, for example, Campbell et al. (1996) Nature, 380:64-68)and Stice et al. (1996) Biol. Reprod., 20 54:100-110). In a particularembodiment, fibroblast cells, such as porcine fibroblast cells can begenetically modified to contain the transgene of interest.

Methods for isolation of oocytes are well known in the art. Essentially,this can comprise isolating oocytes from the ovaries or reproductivetract of a pig. A readily available source of pig oocytes isslaughterhouse materials. For the combination of techniques such asgenetic engineering, nuclear transfer and cloning, oocytes mustgenerally be matured in vitro before these cells can be used asrecipient cells for nuclear transfer, and before they can be fertilizedby the sperm cell to develop into an embryo. This process generallyrequires collecting immature (prophase I) oocytes from mammalianovaries, e.g., bovine ovaries obtained at a slaughterhouse, and maturingthe oocytes in a maturation medium prior to fertilization or enucleationuntil the oocyte attains the metaphase II stage, which in the case ofbovine oocytes generally occurs about 18-24 hours post-aspiration and inthe case of porcine generally occurs at about 35-55 hours. This periodof time is known as the maturation period.”

A metaphase II stage oocyte can be the recipient oocyte, at this stageit is believed that the oocyte can be or is sufficiently “activated” totreat the introduced nucleus as it does a fertilizing sperm. MetaphaseII stage oocytes, which have been matured in vivo have been successfullyused in nuclear transfer techniques. Essentially, mature metaphase IIoocytes can be collected surgically from either non-superovulated orsuperovulated porcine 35 to 48, or 39-41, hours past the onset of estrusor past the injection of human chorionic gonadotropin (hCG) or similarhormone.

After a fixed time maturation period, the oocytes can be enucleated.Prior to enucleation the oocytes can be removed and placed inappropriate medium, such as HECM or TCM199 containing 1 milligram permilliliter of hyaluronidase prior to removal of cumulus cells. Thestripped oocytes can then be screened for polar bodies, and the selectedmetaphase II oocytes, as determined by the presence of polar bodies, arethen used for nuclear transfer. Enucleation follows.

Enucleation can be performed by known methods, such as described in U.S.Pat. No. 4,994,384. For example, metaphase II oocytes can be placed ineither HECM, optionally containing 7-10 micrograms per millilitercytochalasin B, for immediate enucleation, or can be placed in asuitable medium, for example an embryo culture medium such as CR1aa,plus 10% estrus cow serum, and then enucleated later, for example notmore than 24 hours later or 16-18 hours later.

Enucleation can be accomplished microsurgically using a micropipette toremove the polar body and the adjacent cytoplasm. The oocytes can thenbe screened to identify those of which have been successfullyenucleated. One way to screen the oocytes is to stain the oocytes with3-10 microgram per milliliter 33342 Hoechst dye in suitable holdingmedium, and then view the oocytes under ultraviolet irradiation for lessthan 10 seconds. The oocytes that have been successfully enucleated canthen be placed in a suitable culture medium, for example, CR1aa plus 10%serum.

A single mammalian cell of the same species as the enucleated oocyte canthen be transferred into the perivitelline space of the enucleatedoocyte used to produce the NT unit. The mammalian cell and theenucleated oocyte can be used to produce NT units according to methodsknown in the art. For example, the cells can be fused by electrofusion.Electrofusion is accomplished by providing a pulse of electricity thatis sufficient to cause a transient breakdown of the plasma membrane.This breakdown of the plasma membrane is very short because the membranereforms rapidly. Thus, if two adjacent membranes are induced tobreakdown and upon reformation the lipid bilayers intermingle, smallchannels can open between the two cells. Due to the thermodynamicinstability of such a small opening, it enlarges until the two cellsbecome one. See, for example, U.S. Pat. No. 4,997,384 by Prather et al.A variety of electrofusion media can be used including, for example,sucrose, mannitol, sorbitol and phosphate buffered solution. Forexample, the fusion media can comprise a 280 milli molar (mM) solutionof mannitol, containing 0.05 mM MgCl₂ and 0.001 mM CaCl₂ (Walker et al.,Cloning and Stem Cells. 2002; 4(2):105-12). Fusion can also beaccomplished using Sendai virus as a fusogenic agent (Graham, WisterInot. Symp. Monogr., 9, 19, 1969). Also, the nucleus can be injecteddirectly into the oocyte rather than using electroporation fusion. See,for example, Collas and Barnes, (1994) Mol. Reprod. Dev., 38:264-267.After fusion, the resultant fused NT units are then placed in a suitablemedium until activation, for example, CR1aa medium. Typically activationcan be effected shortly thereafter, for example less than 24 hourslater, or about 4-9 hours later for bovine NT and 1-4 hours later forporcine NT.

The NT unit can be activated by known methods. Such methods include, forexample, culturing the NT unit at sub-physiological temperature, inessence by applying a cold, or actually cool temperature shock to the NTunit. This can be most conveniently done by culturing the NT unit atroom temperature, which is cold relative to the physiologicaltemperature conditions to which embryos are normally exposed.Alternatively, activation can be achieved by application of knownactivation agents. For example, penetration of oocytes by sperm duringfertilization has been shown to activate prelusion oocytes to yieldgreater numbers of viable pregnancies and multiple genetically identicalcalves after nuclear transfer. Also, treatments such as electrical andchemical shock can be used to activate NT embryos after fusion. See, forexample, U.S. Pat. No. 5,496,720 to Susko-Parrish et al. Additionally,activation can be effected by simultaneously or sequentially byincreasing levels of divalent cations in the oocyte, and reducingphosphorylation of cellular proteins in the oocyte. This can generallybe effected by introducing divalent cations into the oocyte cytoplasm,e.g., magnesium, strontium, barium or calcium, e.g., in the form of anionophore. Other methods of increasing divalent cation levels includethe use of electric shock, treatment with ethanol and treatment withcaged chelators. Phosphorylation can be reduced by known methods, forexample, by the addition of kinase inhibitors, e.g., serine-threoninekinase inhibitors, such as 6-dimethyl-aminopurine, staurosporine,2-aminopurine, and sphingosine. Alternatively, phosphorylation ofcellular proteins can be inhibited by introduction of a phosphatase intothe oocyte, e.g., phosphatase 2A and phosphatase 2B.

The activated NT units can then be cultured until they reach a suitablesize for transferring to a recipient female, or alternately, they may beimmediately transferred to a recipient female. Culture media suitablefor culturing and maturation of embryos are well known in the art.Examples of known media, which can be used for embryo culture andmaintenance, include Ham's F-10+10% fetal calf serum (FCS), TissueCulture Medium-199 (TCM-199)+10% fetal calf serum,Tyrodes-Albumin-Lactate-Pyruvate (TALP), Dulbecco's Phosphate BufferedSaline (PBS), Eagle's Whitten's media, PZM, NCSU23 and NCSU37. SeeYoshioka K, Suzuki C, Tanaka A, Anas I M, Iwamura S. Biol Reprod. (2002)January; 66(1):112-9 and Petters R M, Wells K D. J Reprod Feral Suppl.1993; 48:61-73.

Afterward, the cultured NT unit or units can be washed and then placedin a suitable media contained in well plates which can optionallycontain a suitable confluent feeder layer. Suitable feeder layersinclude, by way of example, fibroblasts and epithelial cells. The NTunits are cultured on the feeder layer until the NT units reach a sizesuitable for transferring to a recipient female, or for obtaining cellswhich can be used to produce cell colonies. NT units can be cultureduntil at least about 2 to 400 cells, about 4 to 128 cells, or at leastabout 50 cells. Alternatively, NT units may be immediately transferredto a recipient female.

The methods for embryo transfer and recipient animal management in thepresent invention are standard procedures used in the embryo transferindustry. Synchronous transfers are important for success of the presentinvention, i.e., the stage of the NT embryo is in synchrony with theestrus cycle of the recipient female. See, for example, Siedel, G. E.,Jr. (1981) “Critical review of embryo transfer procedures with cattle inFertilization and Embryonic Development in Vitro, L. Mastroianni, Jr.and J. D. Biggers, ed., Plenum Press, New York, N.Y., page 323. Porcineembryo transfer can be conducted according to methods known in the art.For reference, see Youngs et al. “Factors Influencing the Success ofEmbryo Transfer in the Pig,” Theriogenology (2002) 56: 1311-1320.

Production of Multi-Transgenic Animals Containing Endothelial Specific(Endo) Transgenes

Animals (or fetuses) of the invention can be produced according to thefollowing means, including, but not limited to the group selected from:nuclear transfer (NT), natural breeding, rederivation via NT using cellsfrom an existing cell line, fetus, or animal as nucleardonors—optionally adding additional transgenes to these cells prior toNT, sequential nuclear transfer, artificial reproductive technologies(ART) or any combination of these methods or other methods known in theart. In general, “breeding” or “bred” refers to any means ofreproduction, including both natural and artificial means. Further, thepresent invention provides for all progeny of animals produced by themethods disclosed herein. It is understood that in certain embodimentssuch progeny can become homozygous for the genes described herein.

In one embodiment, cells are isolated from animals which lack expressionof GT (GTKO) and are transgenic for CD46 (GTKO/CD46). These cells arefurther modified with an endothelial specific TM transgene, and theresulting transgenic cells are used as nuclear donors to generateGTKO/CD46/TM transgenic animals via NT.

In another embodiment, GTKO/CD46 cells are further modified with anendothelial specific CD39 transgene, and the resulting transgenic cellsare used as nuclear donors to generate GTKO/CD46/CD39 transgenic animalsvia NT.

In a further embodiment, GTKO/CD46 cells are further modified with anendothelial specific EPCR transgene, and resulting transgenic cells areused as nuclear donors to generate GTKO/CD46/EPCR transgenic animals viaNT.

In a further embodiment, GTKO/CD46 cells are further modified withendothelial specific TM and EPCR transgenes, and resulting transgeniccells are used as nuclear donors to generate GTKO/CD46/TM/EPCRtransgenic animals via NT.

In another embodiment, GTKO/CD46/TM animals are mated withGTKO/CD46/CD39 animals to generate GTKO/CD46/TM/CD39 animals viabreeding.

In one embodiment, cells are isolated from animals which lack expressionof GT (GTKO) and are also transgenic for CD46 and DAF (constitutiveexpression). These GTKO/CD46/DAF transgenic cells are further modifiedwith one or more endothelial specific transgenes (ESTR), such ESTRinclude but are not limited to the anticoagulant, immunosuppressantand/or cytoprotective transgenes described herein, and the resultingtransgenic cells are used as nuclear donors to generateGTKO/CD46/DAF/ESTR transgenic animals via NT.

In another embodiment, cells are isolated from animals which lackexpression of GT (GTKO) and are also transgenic for CD46 and CIITA(constitutive expression). These GTKO/CD46/CIITA transgenic cells arefurther modified with one or more endothelial specific transgenes(ESTR), such ESTR include but are not limited to the anticoagulant,immunosuppressant and/or cytoprotective transgenes described herein, andthe resulting transgenic cells are used as nuclear donors to generateGTKO/CD46/CIITA/ESTR transgenic animals via NT.

In a further embodiment, cells are isolated from animals which lackexpression of GT (GTKO) and are transgenic for CD46, DAF and CIITA(constitutive expression). These GTKO/CD46/DAF/CIITA cells are furthermodified with one or more endothelial specific transgenes (ESTR), suchESTR include but are not limited to the anticoagulant, immunosuppressantand/or cytoprotective transgenes described herein, and resultingtransgenic cells are used as nuclear donors in NT to generateGTKO/CD46/DAF/CIITA ESTR transgenic animals.

In a further embodiment, GTKO/CD46/DAF/CIITA animals are bred toGTKO/CD46/endo transgenic animals to generate GTKO/CD46/DAF/CIITA ESTRtransgenic animals

In a further embodiment, GTKO/CD46/TM animals are bred toGTKO/CD46/DAF/CIITA transgenic animals to generateGTKO/CD46/TM/DAF/CIITA animals.

In a further embodiment, GTKO/CD46 animals which additionally contain anendothelial specific transgene are bred to GTKO/CD46/DAF/CIITAtransgenic animals to generate GTKO/CD46/DAF/CIITA/ESTR transgenicanimals via breeding.

In another embodiment, cells isolated from GTKO/CD46/TM animals arefurther modified with an immunosuppressant transgene, such as pCTLA4Ig.The resulting transgenic cells are used as nuclear donors to generateGTKO/CD46/TM/CTLA4Ig animals via NT.

In certain embodiments cells isolated from GTKO/CD46/TM animals arefurther modified with one or more immunomodulatory or anticoagulanttransgenes, and the resulting cells containing four or more transgenesare used as nuclear donors to generate multi-transgenic animals via NT.

In further embodiments, any of the multitransgenic animals embodiedherein can be bred together naturally, or using artificial reproductivetechnologies to generate multi-transgenic animals with additionalgenetic modifications via breeding.

In addition, cells isolated from any of the multitransgenic animals (orfetuses) embodied herein can be used in further NT to rederive animals,or to add further genetic modifications to their genome followed by NTto generate multi-transgenic animals containing additional geneticmodifications via NT.

Whole Organ Xenografts

There is a critical shortage of human organs for the purposes of organtransplantation. In the United States alone approximately 110,000patients are on waiting lists to receive organs, and yet only 30,000organs will become available from deceased donors. Almost 20 patientsdie each day (7,000 per year) waiting for an organ (Cooper and Ayares,2010 International Journal of Surgery, In Press,doi:10.1016/j.ijsu.2010.11.002). The supply of human organs for use inallotransplantion will never fully meet the population's need. A newsource of donor organs is urgently needed.

Xenotransplantation could effectively address the shortage of humandonor organs. Xenotransplants are also advantageously (i) supplied on apredictable, non-emergency basis; (ii) produced in a controlledenvironment; and (iii) available for characterization and study prior totransplant.

Depending on the relationship between donor and recipient species, thexenotransplant can be described as concordant or discordant. Concordantspecies are phylogenetically closely related species (e.g., mouse torat). Discordant species are not closely related (e.g., pig to human).Pigs have been the focus of most research in the xenotransplanationarea, since the pig shares many anatomical and physiologicalcharacteristics with human. Pigs also have relatively short gestationperiods, can be bred in pathogen-free environments and may not presentthe same ethical issues associated with animals not commonly used asfood sources (e.g., primates). The transplantation of whole porcineorgans into non-human primates has been reviewed (see for example Ekseret al., Transplant Immun. 2009 21:87-92; Ekser and Cooper. Expert RevClin Immunol. 2010 March; 6(2):219-30; Mohiuddin, M. PLoS Med. 2007 Mar.27; 4(3):e75; Pierson et al., Xenotransplantation. 2009September-October; 16(5):263-80). For therapeutic use of porcine organsto become available for use in human medical treatment, improvedoutcomes must first be obtained in non-human primate pre-clinicaltrials, followed by duplication or improvement of these results in humanclinical trials. The pigs of the current invention can provide a sourceof porcine donor organs to address these requirements.

In additional embodiments, organs according to the present invention canbe selected from the following: heart, lung, liver, kidney, intestine,spleen, and pancreas. In one embodiment, the xenotransplanted organs ofthe present invention can survive and function in the recipient like anallograft. In other embodiments, the organs described herein can be usedas bridge organs until a human organ becomes available. In oneembodiment, the bridge organ can be used in a recipient for at least 3days. In other embodiments, the bridge organ can be used in a recipienta period of time selected from the following: at least 4, 5, 6, 7, 8, 9,10, 14, 21, 28 days.

Hearts

In one embodiment, hearts obtained from animals of the current inventioncan be used pre-clinically and clinically to improve outcomes in cardiacxenotransplantation. Heart transplants can be heterotopic(non-life-supporting: the endogenous organ remains in place) ororthotopic (life-supporting, where the heart is replaced with a donorheart). In one embodiment, the heart transplants can be heterotropic. Inanother embodiment, the heart transplants can be orthotropic. Innon-human primate xenotransplant studies, the majority to date has beenheterotopic grafts, but in later studies and in human clinical use,hearts will be transplanted orthotopically.

In one embodiment, hearts from pigs of the invention, when transplantedinto a primate, can function for at least six months in a majority ofprimates. The majority of primates can be at least 70%, at least 75%, atleast 80% or at least 90% of primtated. In other embodiments, thetransplanted hearts of the present invention can function for a timeperiod of at least 8 months, at least 9 months, at least 10 months, atleast 11 months, at least 12 months, at least 15 months, at least 18months, at least 21 month, at least 24 months, at least 36 months, or atleast 48 months. Such transplants can be heterotopic or orthotopic.

In one embodiment, hearts from the pigs of the invention, whenorthotopically transplanted in to a human can function for up to 9months.

Using GTKO pigs and novel immunosuppressant agents, 2 to 6 months'survival of heterotopic heart xenotransplants was achieved (Kuwaki etal. Nat Med 2005:11:29-31; Tseng et al, Transplantation2005:80:1493-500). Transgenic pigs with the combination of GTKO andexpression of CD46 were recently tested in a heterotopic heart model(pig-to-baboon) and provided prolonged survival and function ofxenograft hearts for up to 8 months. (Mohiuddin et al., AbstractTTS-1383. Transplantation 2010; 90 (suppl): 325).

In non-human primate heart xenotransplant studies, graft failure hasoccurred due to the development of a thrombotic microangiopathy thatresults in vascular occlusion and surrounding ischemic injury. Heartsfrom the pigs of the current invention, which express anticoagulanttransgenes in the vascular endothelium will lesson or prevent thromboticevents, such as, for example, consumptive coagulopathy and thromboticmicroangiopathy, from occurring and serve to protect the xenograft frominjury. In one embodiment, hearts from the pigs disclosed herein candecrease thrombotic events. In another embodiment, hearts from the pigsdisclosed herein can prevent thrombotic events. For reviews of progressin this heart xenotransplantation field over the past 20 years pleasesee for example, Zhu et al, J Heart Lung Transplant. 2007 March;26(3):210-8 and Ekser and Cooper, Curr Opin Organ Transplant. 2008October; 13(5):531-5. The use of porcine donor hearts as a bridgetransplant has also been detailed (Cooper and Teuteberg J Heart LungTransplant. 2010 August; 29(8):838-40).

Additional embodiments encompass, pigs of the current inventioncontaining further genetic modifications, for example, immunosuppressanttransgenes, for example, endothelial expression of immunosuppressanttransgenes, such as CTLA4-Ig, allows for the use of a clinicallyrelevant immunosuppressant regimen to be used in cardiacxenotransplantation.

In other embodiments, the porcine heart can be transferred to a primateand can function in the primate for at least 6 months. In anotherembodiment, the porcine heart can be transferred to a primate and canfunction in the primate for at least 9 months. In a further embodiment,the porcine heart can be transferred to a primate and can function inthe primate for at least 12 months. In a still further embodiment, theporcine heart can be transferred to a primate and can function in theprimate for at least 18 months. In certain embodiments, the primate canbe a monkey. In another embodiment, the primate can be a baboon. In afurther embodiment, the primate can be a human. In one embodiment, atleast 6 primates can be tested with the porcine heart. In anotherembodiment, at least 8 primates can be tested with the porcine heart. Ina further embodiment, at least 10 primates can be tested with theporcine heart.

In one embodiment, hearts from the pigs of the invention, whentransplanted into a primate can serve as a bridge to an allotransplant.In a specific embodiment, the hearts can be used as bridge organs for atime selected form but not limited to at least 1 month, at least 2months or at least 3 months. In one specific embodiment, the porcineheart can be used as a bridge transplant and function in the primate forat least 9 months, at least 12 months or at least 15 months. In oneembodiment, the primate can be a non-human primate. In anotherembodiment, the primate can be human.

For details on the transplantation procedure, see, for example, Handbookof Animal Models in Transplantation Research, Edited by D. V. Cramer, L.Podesta, L. Makowka 1994 CRC Press, for example, Chapters 3, 7, 8, 9 and14; Cooper et al “Report of the Xenotransplantation Advisory Committeeof the International Society for Heart and Lung Transplantation”December 2000 The Journal of Heart and Lung Transplantation, pp1125-1165.

Kidneys

The use of GTKO pigs and/or transgenic pigs overexpressing humancomplement inhibitor genes for kidney xenotransplantation has largelyovercome the problem of HAR, however problems remain with xeno-kidneysbeing rejected via AHXR. Yamada et al, (Nat Med. 2005 January;11(1):32-4) obtained survival of >80 days in two baboons. Histology ofmany of the kidneys showed preserved structure, but the relativelyintensive immunosuppressive regimen required to prolong graft survivalresulted in complications. Less encouraging data in the GT-KOpig-to-baboon model were reported by Chen et al (Nat Med 2005:11:1295-8)where in contrast to the studies of Yamada et al., an elicitedanti-nonGal antibody response was not prevented and AHXR resulted ingraft failure.

Kidneys from the multi-transgenic pigs of the invention can decrease oreliminate xenorejection, exhibiting improved outcomes when used as adiscordant transplant. In one embodiment, kidneys from the pigs of theinvention remain functional in a non-human primate and do not exhibitxenorejection for 6 months or more. In another embodiment, kidneys fromthe pigs of the invention remain functional in a human for a year ormore. In other embodiments, the transplanted kidneys of the presentinvention can function for a time period of at least 8 months, at least9 months, at least 10 months, at least 11 months, at least 12 months, atleast 15 months, at least 18 months, at least 21 month, at least 24months, at least 36 months, or at least 48 months. Such transplants canbe heterotopic or orthotopic.

Additionally, kidneys from pigs of the current invention containingfurther genetic modifications, for example, immunosuppressant transgenessuch as CTLA4-Ig, will allow for clinically acceptable levelsimmunosuppression to be used, leading to fewer complications as a resultof treatment.

Additional embodiments encompass, pigs of the current inventioncontaining further genetic modifications, for example, immunosuppressanttransgenes, for example, endothelial expression of immunosuppressanttransgenes, such as CTLA4-Ig, allows for the use of a clinicallyrelevant immunosuppressant regimen to be used in renalxenotransplantation.

In one embodiment, the porcine kidney can be transferred to a primateand can function in the primate for at least 6 months. In anotherembodiment, the porcine kidney can be transferred to a primate and canfunction in the primate for at least 9 months. In a further embodiment,the porcine kidney can be transferred to a primate and can function inthe primate for at least 12 months. In a still further embodiment, theporcine kidney can be transferred to a primate and can function in theprimate for at least 18 months. In certain embodiments, the primate canbe a monkey. In another embodiment, the primate can be a baboon. In afurther embodiment, the primate can be a human. In one embodiment, atleast 6 primates can be tested with the porcine kidney. In anotherembodiment, at least 8 primates can be tested with the porcine kidney.In a further embodiment, at least 10 primates can be tested with theporcine kidney. In one specific embodiment, the porcine kidney can beused as a bridge transplant and function in the primate for at least 9months. In another specific embodiment, the porcine kidney can be usedas a bridge transplant and function in the primate for at least 12months. In a further specific embodiment, the porcine kidney can be usedas a bridge transplant and function in the primate for at least 15months.

For details on the transplantation procedure, see, for example, Handbookof Animal Models in Transplantation Research, Edited by D. V. Cramer, L.Podesta, L. Makowka 1994 CRC Press, for example, Chapters 3, 7, 8, 9 and14.

Pancreas

In certain embodiments, the pancreas from the multi-transgenic pigs ofthe invention can be used. Such pancreas can decrease or eliminatexenorejection, exhibiting improved outcomes when used as a discordanttransplant.

In a further embodiment a kidney xenotransplant using kidneys from thepigs of the invention can be combined with a pancreas or pancreaticislet transplant. For example, this is currently performed inallotransplantation to treat patients with type 1 diabetes and latechronic kidney disease (reviewed by Wiseman, Curr Diab Rep. 2010October; 10(5):385-91; Adv Chronic Kidney Dis. 2009 July; 16(4):278-87).

In one embodiment, the porcine pancreas can be transferred to a primateand can function in the primate for at least 6 months. In anotherembodiment, the porcine pancreas can be transferred to a primate and canfunction in the primate for at least 9 months. In a further embodiment,the porcine pancreas can be transferred to a primate and can function inthe primate for at least 12 months. In a still further embodiment, theporcine pancreas can be transferred to a primate and can function in theprimate for at least 18 months. In certain embodiments, the primate canbe a monkey. In another embodiment, the primate can be a baboon. In afurther embodiment, the primate can be a human. In one embodiment, atleast 6 primates can be tested with the porcine pancreas. In anotherembodiment, at least 8 primates can be tested with the porcine pancreas.In a further embodiment, at least 10 primates can be tested with theporcine pancreas. In one specific embodiment, the porcine pancreas canbe used as a bridge transplant and function in the primate for at least9 months. In another specific embodiment, the porcine pancreas can beused as a bridge transplant and function in the primate for at least 12months. In a further specific embodiment, the porcine pancreas can beused as a bridge transplant and function in the primate for at least 15months. In alternate embodiments, the uses of the porcine pancreasdisclosed herein can be used in combination with a kidney transplant.

Additional embodiments encompass, pigs of the current inventioncontaining further genetic modifications, for example, immunosuppressanttransgenes, for example, endothelial expression of immunosuppressanttransgenes, such as CTLA4-Ig, allows for the use of a clinicallyrelevant immunosuppressant regimen to be used in pancreatic/renalxenotransplantation.

For details on the transplantation procedure, see, for example, Handbookof Animal Models in Transplantation Research, Edited by D. V. Cramer, L.Podesta, L. Makowka 1994 CRC Press, for example, Chapters 3, 7, 8, 9 and14.

Lungs

Xenotransplantation of porcine lungs is briefly reviewed in Ekser etal., Transplant Immun. 2009 21:87-92, but there is little dataavailable. Lungs from the pigs of the current invention, will allow forfurther pre-clinical and clinical progress to be made. The transplantedlungs of the current invention can be a full lung or lung pair.

In embodiments of the present invention, the transplanted lungs of thepresent invention can function for a time period of at least 1 month, atleast 2 months, at least 3 months, at least 4 months, at least 6 months,at least 8 months, at least 9 months, at least 10 months, at least 11months, at least 12 months, at least 15 months, at least 18 months, atleast 21 month, at least 24 months, at least 36 months, or at least 48months. Such transplants can be heterotopic or orthotopic.

In other embodiments, the porcine lung can be transferred to a primateand can function in the primate for at least 6 months. In anotherembodiment, the porcine lung can be transferred to a primate and canfunction in the primate for at least 9 months. In a further embodiment,the porcine lung can be transferred to a primate and can function in theprimate for at least 12 months. In a still further embodiment, theporcine lung can be transferred to a primate and can function in theprimate for at least 18 months. In certain embodiments, the primate canbe a monkey. In another embodiment, the primate can be a baboon. In afurther embodiment, the primate can be a human. In one embodiment, atleast 6 primates can be tested with the porcine lung. In anotherembodiment, at least 8 primates can be tested with the porcine lung. Ina further embodiment, at least 10 primates can be tested with theporcine lung.

In one embodiment, lungs from the pigs of the invention, whentransplanted into a primate can serve as a bridge to an allotransplant.In a specific embodiment, the lungs can be used as bridge organs for atime selected form but not limited to at least 7 days, at least 14 days,at least 21 days or at least 1 months. In one specific embodiment, theporcine lung can be used as a bridge transplant and function in theprimate for at least 3 months, at least 4 months or at least 6 months.In another embodiment, the lung can be used as a bridge organ for 3-6months. In one embodiment, the primate can be a non-human primate. Inanother embodiment, the primate can be human.

In particular embodiment, lungs are provided from transgenic animalsthat lack any expression of functional alpha 1,3 galactosyltransferase(GTKO) and specifically expresses at least one transgene in endothelialtissue. In another embodiment, lungs are provided from transgenicanimals that lack any expression of functional alpha 1,3galactosyltransferase (GTKO) and expresses at least one complimentinhibitor and specifically expresses at least one transgene inendothelial tissue selected from the group consisting ofanti-coagulants, immunomodulators and cytoprotectants. In a specificembodiment, lungs are provided from transgenic animals with thefollowing genetic modifications: GTKO, ubiquitous expression of at leastone complement inhibitor, endothelial specific expression of at leastthree anticoagulants, and at least one immunomodulators. In anadditional embodiment, the animal can also express at least onecytoprotective element. In a particularly specific embodiment, a lungfrom a transgenic animal is provided wherein the animal has thefollowing genetic modifications: GTKO, DAF, CD46, andendothelial-specific expression of CD39, TM, EPCR, TFPI, CIITA-DN. In afurther embodiment, the animal can also express A20 and HO-1.

Additional embodiments encompass, pigs of the current inventioncontaining further genetic modifications, for example, immunosuppressanttransgenes, for example, endothelial expression of immunosuppressanttransgenes, such as CTLA4-Ig, allows for the use of a clinicallyrelevant immunosuppressant regimen to be used in pulmonaryxenotransplantation.

For details on the transplantation procedure, see, for example, Handbookof Animal Models in Transplantation Research, Edited by D. V. Cramer, L.Podesta, L. Makowka 1994 CRC Press, for example, Chapters 3, 7, 8, 9 and14; Cooper et al “Report of the Xenotransplantation Advisory Committeeof the International Society for Heart and Lung Transplantation”December 2000 The Journal of Heart and Lung Transplantation, pp1125-1165.

Livers

The use of porcine livers in xenotransplantation has been reviewed byHara (Liver Transpl. 2008 April; 14(4):425-34) and porcine livers fromGTKO/CD46 pigs have recently shown parameters of liver function in thenear-normal range (Ekser et al, Transplantation. 2010 Sep. 15;90(5):483-93). In the human clinic, porcine livers are most likely to beused as a bridge transplant until a human derived liver becomesavailable for transplant. This use of livers from the pigs of theinvention is detailed in the next section.

In other embodiments, the transplanted livers of the present inventioncan function for a time period of at least 3 months, at least 6 months,8 months, at least 9 months, at least 10 months, at least 11 months, atleast 12 months, at least 15 months, at least 18 months, at least 21month, at least 24 months, at least 36 months, or at least 48 months.Such transplants can be heterotopic or orthotopic. In anotherembodiment, the livers of the present invention can be used as a bridgetransplant.

In one specific embodiment, the porcine liver can be used as a bridgetransplant and function in the primate for at least 2 weeks, at least 3weeks, or at least 4 weeks. In another specific embodiment, the porcineliver can be used as a bridge transplant and function in the primate forat least 2 weeks. In a further specific embodiment, the porcine livercan be used as a bridge transplant and function in the primate for atleast 6 weeks. In another specific embodiment, the porcine liver can beused as a bridge transplant and function in the primate for at least 8weeks. In a further specific embodiment, the porcine liver can be usedas a bridge transplant and function in the primate for at least 12weeks.

Additional embodiments encompass, pigs of the current inventioncontaining further genetic modifications, for example, immunosuppressanttransgenes, for example, endothelial expression of immunosuppressanttransgenes, such as CTLA4-Ig, allows for the use of a clinicallyrelevant immunosuppressant regimen to be used in hepaticxenotransplantation.

For details on the transplantation procedure, see, for example, Handbookof Animal Models in Transplantation Research, Edited by D. V. Cramer, L.Podesta, L. Makowka 1994 CRC Press, for example, Chapters 3, 7, 8, 9 and14

Other Xenograft Applications

In addition to their use for therapeutic whole organ replacement, theporcine organs tissues and cells of the invention have additionaltherapeutic applications.

For example, porcine livers of the invention can be used as a bridge toan allo-transplant. The use of pig-livers for bridge transplants isreviewed in depth by Ekser et al. (2009. Transplantation. Nov. 15 88(9):1041-1049). In embodiments of the invention, porcine liver xenograftscan function and serve to stabilize a patient undergoing liver failurefor at least 5 days, at least 7 days, at least 14 days at least 21 daysor at least 30 days. Such transplants can be used as a bridge until asuitable allotransplant liver becomes available.

Porcine liver tissues and cells of the invention can also be used inbioartifical liver (BAL) devices. BAL devices are used to providetemporary liver support to bridge a patient with rapidly deterioratingliver function to orthotopic liver transplant or to allow time for liverregeneration. There are several groups developing bioartificial liverdevices, for example, Circe Biomedical (Lexington, Mass.), Vitagen (LaJolla, Calif.), Excorp Medical (Oakdale, Minn.), and Algenix (Shoreview,Minn.). The Circe Biomedical device integrates viable liver cells withbiocompatible membranes into an extracorporeal, bioartificial liverassist system. Formerly developed by Circe and Arbios, this technology,HepaMate™, is now being developed by Hepalife(http://www.hepalifebiosystems.com/clinical-trials.php). Vitagen's ELAD(Extracorporeal Liver Assist Device) Artificial Liver is a two-chamberedhollow-fiber cartridge containing a cultured human liver cell line(C3A). The cartridge contains a semipermeable membrane with acharacterized molecular weight cutoff: This membrane allows for physicalcompartmentalization of the cultured human cell line and the patient'sultrafiltrate. Algenix provides a system in which an external liversupport system uses porcine liver cells. Individual porcine hepatocytespass through a membrane to process the human blood cells. ExcorpMedical's device contains a hollow fiber membrane (with 100 kDa cutoff)bioreactor that separates the patient's blood from approximately 100grams of primary porcine hepatocytes that have been harvested from,purpose-raised, pathogen-free pigs. Blood passes though a cylinderfilled with hollow polymer fibers and a suspension containing billionsof pig liver cells. The fibers act as a barrier to prevent proteins andcell byproducts of the pig cells from directly contacting the patient'sblood but allow the necessary contact between the cells so that thetoxins in the blood can be removed. In certain embodiments the porcinecells of the invention, used in a BAL device, can function and serve tostabilize a patient undergoing liver failure for up to 7 days, up to 14days, or up to 30 days until a suitable allotransplant liver becomesavailable. Other uses of porcine livers, tissues or cells of theinvention, including in extracorporeal artificial liver devices, inextracorporeal liver perfusion procedures, and for hepatic celltransplantation, as a bridge to an orthotopic allotransplant, or tosupport patient liver function and regeneration (as detailed forexample, in Ekser et al. 2009. Transplantation. Nov. 15 88(9):1041-1049) are also embodied herein.

Endothelial cells isolated from the cornea of animals of the inventioncan be used as a graft to treat cornea dysfunction. Optical surgicaltransplant procedures known as endothelial keratoplasty (EK) replacedysfunctional cornea endothelium with donor material. A procedure knownas Deep Lamellar Endothelial Keratoplasty (DLEK) has become widely usedsince its introduction (Terry, M. A., Cataract and Refractory SurgeryToday February 2004, p. 1-3). For a current review of the various EKprocedures see Melles, September 2006 Cornea Volume 25(8):879-881.

Endothelial cells isolated from the retina of animals of the inventioncan be used as a graft to treat retina dysfunction, to treat diseasesincluding acute macular degeneration or diabetes induced retinopathy. Incertain embodiments, retinal endothelial cells can be used in thetreatment of retinal dysfunction. In another embodiment, retinalendothelial cells can be used in the treatment of acute maculardegeneration. In another embodiment, retinal endothelial cells can beused in the treatment of diabetes induced retinopathy.

Porcine tissues and cells from the animals of the invention can be usedas vascular grafts. The current clinical source of vascular graftmaterials is limited to: vessels taken from the patient (autologous),tissue banks (allograft), materials derived from animals and highlyprocessed to remove antigens and viable cells, or synthetic materials.There have also been efforts to develop bio-engineered vascular graftmaterials, however, challenges remain in this newly developing field,and such grafts are not yet clinically available (Campbell and Campbell,Curr Pharm Biotechnol. 2007 February; 8(1):43-50). For an extensivereview of existing vascular graft materials and their applications, seefor example Leon L, and Greisler H P. Expert Rev Cardiovasc Ther. 2003November; 1(4):581-94. While autologous grafting is the method ofchoice, many patients do not have suitable vessels available forautologous grafting. Human derived allografts from tissue banks presentrisk of disease transmission to the recipient (Eastlund T. CellTransplant. 1995 September-October; 4(5):455-77). Highly processedanimal materials have shown problems with durability and immunogenicity(Lehalle B, Geschier C, Fiévé G, Stoltz J F. J Vasc Surg. 1997 April;25(4):751-2).

Vascular tissues and cells from the animals of the invention can providea safe alternate supply of vascular grafts. In certain embodiments ofthe present invention, vascular grafts can be selected from the groupincluding, heart valves, femoral vein, femoral artery, aortoiliacartery, saphenous vein, ascending aorta, pulmonary artery, thoracicaorta, pulmonary artery, internal mammary artery, radial artery, or anyother vessel that is currently used therapeutically as an autologousgraft or allograft. In one embodiment, a single valved section of mainpulmonary trunk may be used as a mono-cusp patch(www.AccessLifeNetHealth.org). In other embodiments, vascular materialsfrom the animals of the invention can be used for replacement, shunting,patching or repair to treat a vascular defect or disease.

In further embodiments, vascular grafts from the animals of theinvention can be used for vascular reconstructive surgery, coronarybypass surgery, or arterial or venous grafting. In certain embodiments,vascular grafts described herein can be used to treat a disease selectedfrom the group including but not limited to atherosclerosis, coronaryartery disease, peripheral vascular disease, and aortic aneurysm. Inother embodiments, the vascular grafts disclosed herein can be used forperipheral vascular bypass surgery. In certain embodiments, the graftsdisclosed herein can be used to treat peripheral arterial disease,critical limb ischemia or any other vascular occlusion.

In further embodiments, the porcine endothelial cells of the inventioncan also be used to seed vascular grafts, or can be used for seedingduring coronary procedures, such as stenting or bypass surgery. Vasculargraft materials can be allografts (human origin), or bioengineereddevices, or any other material used as a vascular graft. Details on theuse of endothelial cells for seeding following coronary procedures canbe found for example in Kipshidze et al., J. Am. Coll. Cardiol. 2004;44; 733-739 and details on the construction of vascular grafts andendothelial cell seeding methods can be found for example in Sarkar etal., J Biomed Mater Res B Appl Biomater. 2007 July; 82(1):100-8 andVillalona et al., Tissue Eng Part B Rev. 2010 June; 16(3):341-50. For arecent review of vascular engineered biomaterials (including xenograftmaterials) see Ravi and Chaikof, Regen Med. 2010 January; 5(1):107-20.

The methods of the invention also include methods of xenotransplantationwherein the transgenic organs, tissues or cells provided herein aretransplanted into a primate and, after the transplant, the primaterequires minimal or no immunosuppressive therapy. Reduced or noimmunosuppressive therapy includes, but is not limited to, a reduction(or complete elimination of) in dose of the immunosuppressivedrug(s)/agent(s) compared to that required by other methods; a reduction(or complete elimination of) in the number of types of immunosuppressivedrug(s)/agent(s) compared to that required by other methods; a reduction(or complete elimination of) in the duration of immunosuppressiontreatment compared to that required by other methods; and/or a reduction(or complete elimination of) in maintenance immunosuppression comparedto that required by other methods.

Additional embodiments encompass, pigs of the current invention allowsfor the use of a clinically relevant immunosuppressant regimen to beused in pulmonary xenotransplantation.

Further embodiments encompass, pigs of the current invention containinggenetic modifications of the present invention allows for the use of aclinically relevant immunosuppressant regimen to be used inxenotransplantation. For example, immunosuppressant transgenes can beused. In one embodiment, endothelial expression of immunosuppressanttransgenes can be used. The immunosuppressant transgene can be CTLA4-Ig.

The methods of the invention also include methods of treating orpreventing organ dysfunction wherein after the transplantation oftransgenic organs, tissues or cells, the transplant is repeated. Thetransplant may be performed twice, three times or more in any oneprimate. The transplant may occur once a year. The transplant may occurtwice a year. The transplant may occur three times a year. Thetransplant may occur more than three times a year. The transplant mayoccur at various times over multiple years. The parameters of any onetransplant, including, but not limited to, surgical procedures, deliverymethods, donor tissues and/or cells used, immunosuppressive regimensused and the like, may be different or the same when compared to othertransplants performed in the same primate.

In some embodiments, the method reduces the need for administration ofanti-inflammatories to the host. In other embodiments, the methodreduces the need for administration of anticoagulant to the host. Incertain embodiments, the method reduces the need for administration ofimmunosuppressive agents to the host. In some embodiments, the host isadministered an anti-inflammatory agent for less than thirty days, orless than 20 days, or less than 10 days, or less than 5 days, or lessthan 4 days, or less than 3 days, or less than 2 days, or less than oneday after transplantation. In some embodiments, the host is administeredan anticoagulant agent for less than thirty days, or less than 20 days,or less than 10 days, or less than 5 days, or less than 4 days, or lessthan 3 days, or less than 2 days, or less than one day aftertransplantation. In some embodiments, the host is administered animmunosuppressive agent for less than thirty days, or less than 20 days,or less than 10 days, or less than 5 days, or less than 4 days, or lessthan 3 days, or less than 2 days, or less than one day aftertransplantation.

The recipient (host) may be partially or fully immunosuppressed or notat all at the time of transplant. Immunosuppressive agents/drugs thatmay be used before, during and/or after the time of transplant are anyknown to one of skill in the art and include, but are not limited to,MMF (mycophenolate mofetil (Cellcept)), ATG (anti-thymocyte globulin),anti-CD154 (CD40L), alemtuzumab (Campath), CTLA4-Ig (Abatacept/Orencia),belatacept (LEA29Y), sirolimus (Rapimune), tacrolimus (Prograf),anti-CD20 (Rituximab), daclizumab (Zenapax), basiliximab (Simulect),infliximab (Remicade), cyclosporine, deoxyspergualin, soluble complementreceptor 1, cobra venom, methylprednisolone, FTY720, everolimus,anti-CD154-Ab, leflunomide, anti-IL-2R-Ab, rapamycin, and humananti-CD154 monoclonal antibody. One or more than one immunosuppressiveagents/drugs may be used together or sequentially. One or more than oneimmunosuppressive agents/drugs may be used for induction therapy or formaintenance therapy. The same or different drugs may be used during theinduction and maintenance stages. In one embodiment, daclizumab(Zenapax) is used for induction therapy and tacrolimus (Prograf) andsirolimus (Rapimune) is used for maintenance therapy. In anotherembodiment, daclizumab (Zenapax) is used for induction therapy and lowdose tacrolimus (Prograf) and low dose sirolimus (Rapimune) is used formaintenance therapy. In one embodiment, alemtuzumab (Campath) is usedfor induction therapy. See Teuteberg et al., Am J Transplantation,10(2):382-388. 2010; van der Windt et al., 2009, Am. J. Transplantation9(12):2716-2726. 2009; Shapiro, The Scientist, 20(5):43. 2006; Shapiroet al., N Engl J Med. 355:1318-1330. 2006. Immunosuppression may also beachieved using non-drug regimens including, but not limited to, wholebody irradiation, thymic irradiation, and full and/or partialsplenectomy. These techniques may also be used in combination with oneor more immunosuppressive drug/agent.

Sufficient time to allow for engraftment (for example, 1 week, 3 weeks,and the like) is provided and successful engraftment is determined usingany technique known to one skilled in the art. These techniques mayinclude, but are not limited to, One or more techniques may be used todetermine if engraftment is successful. Successful engraftment may referto relative to no treatment, or in some embodiments, relative to otherapproaches for transplantation (i.e., engraftment is more successfulthan when using other methods/tissues for transplantation). In somecases, successful engraftment is illustrated by a reduced need forimmunosuppression. This reduced need for immunosuppression may includethe lowering of a dose of one or more immunosuppressive drugs/agents, adecrease in the number of types of immunosuppressive drugs/agentsrequired, a shorter duration of immunosuppression, and/or lower or nomaintenance immunosuppression.

In one embodiment, successful engraftment may be assessed by monitoringor testing for functionality (partial or full) of the transplantedtissue. For heart xenografts this may include, for example, monitoringby palpation, or by continuous telemetry. Progressive bradycardia anddecreasing QRS amplitude are predictive of imminent graft failure(heterotopic abdominal heart xenotransplant; for technique details see,for example, Adams et al., 1999 Ann Thorac Surg. July; 68(1):265-8).Other methods employed by those in the art to monitor cardiac xenograftfunction (in a heterotopic thoracic heart xenotransplant) include, forexample, continuously analyzing heart rate, rhythm and ST-segment inECG-leads II and V5 (Sirecust 960; Siemens, Erlangen, Germany);continuously monitoring arterial blood pressure and cardiac function viaa catheter in the femoral artery (Pulsion, Munich, Germany) and acentral venous catheter (Arrow, Erding, Germany) introduced via thecephalic vein; measuring cardiac output with the femoral arterialthermodilution technique (PiCCO; Pulsion); assessing heart rate of therecipient and the graft by external ECG positioned over the right chestwall daily postoperatively; conducting echocardiographic examinations ofthe graft in regular intervals using an utrasonographic scanner and a10-MHz phased-array transducer (Sonos 5500; Hewlett Packard, Andover,Mass., USA) and performing a CT angiogram (Bauer et al., 2010Xenotransplantation 17:243-249).

EXAMPLES

Generation and Characterization of Multi-Transgenic Pigs withEndothelial Specific Expression, Using Two Different AnticoagulantGenes.

Example 1

Construction of Endothelial Specific Vectors for Production ofTransgenic Pigs

Endothelium-specific expression provides a strategy to limit expressionof bioactive transgenes that could have adverse effects if expressedubiquitously. Two expression systems used (in this example) are theporcine ICAM-2 promoter/enhancer system and the mouse Tie-2promoter/enhancer system.

Examples of anticoagulant transgenes expressed via these endothelialspecific vector systems include:

-   -   1. human CD39 (vector pREV859B, which utilizes the Tie-2        promoter/enhancer and vector pREV861 which utilizes the ICAM-2        promoter/enhancer),    -   2. human thrombomodulin (vector pREV872, which utilizes the        ICAM-2 promoter/enhancer),    -   3. human endothelial protein C receptor (vector pREV873, which        utilizes the ICAM-2 promoter/enhancer),    -   4. human tissue factor pathway inhibitor (vector pREV871, which        utilizes the ICAM-2 promoter/enhancer).

All of these transgenes encode proteins that can inhibit vascularthrombosis during xenotransplantation. These vectors have been shown todrive transgene expression in transiently or stably-transfected porcineendothelial cells. FIG. 1 shows expression analysis of TM and EPCR inimmortalized porcine endothelial cells (IPEC) using flow cytometry.Endothelium specific vectors herein can be used to producemulti-transgenic pigs that exhibit good viability while producingtherapeutic anticoagulants locally within the donor organs, cells, ortissues for support of xenotransplantation.

Vector Construction:

The backbone vector for these constructs contained 5′ and 3′ chickenβ-globin insulators, a multiple cloning site (MCS) and an SV40 polyadenylation signal. Transgene inserts were subcloned into the MCSupstream of the SV40 polyadenylation signal using appropriaterestriction sites described below for each vector.

The pREV859B, Tie-2 CD39 vector was built by insertion of a Nhe1/Sal1fragment containing the Tie-2 enhancer/promoter, and an Xho1 fragmentcontaining the CD39 transgene into the base vector.

The pREV861, ICAM-2 CD39 vector was built by excision of the Tie-2enhancer and promoter in the pREV859B vector with BssHII and BstB1 andinsertion of a BssHII/BstBI fragment containing the ICAM-2 enhancer andpromoter.

The pREV871, ICAM-2 TFPI vector was built by excising the Tie-2enhancer/promoter from a previously built Tie-2 TFPI vector andreplacing it with a BssHII/BstB1 fragment containing the ICAM-2 enhancerand promoter.

The pREV872, ICAM-2 TM vector was built by insertion of an Spe1/Not1ICAM-2 enhancer/promoter fragment, as well as a Not1/Sal1 fragmentcontaining the TM transgene into the base vector.

The pREV873, ICAM-2 EPCR vector was built by insertion of an SpeI/NotIfragment containing the ICAM-2 enhancer/promoter, and a NotI/SpeIfragment containing the EPCR transgene into the base vector.

Example 2

Cell Line Preparation for Nuclear Transfer

Isolation of Cell Lines:

One cell line (183-6-6) was used as the genetic background fortransfections to add the additional transgenes, and ultimately fornuclear transfer to generate pigs. This cell lines was produced bybreeding of GTKO pigs (Dai et al., (2002) Nature biotechnology 20,251-255; Phelps et al., Science, (2003) 299:411-414) with ubiquitouslyexpressing hCD46 transgenic pig lines (Loveland et al.,Xenotransplantation, 2004, 11:171:183). The 186-6-6 cell line wasconfirmed by genotype and phenotype as homozygous GTKO and hCD46transgenic. The cells were prepared for use in NT as follows: A fetalfibroblast cell line was isolated from fetus 183-6-6 at day 36 ofgestation. The Fetus was mashed through a 60-mesh metal screen usingcurved surgical forceps slowly so as not to generate excessive heat. Thecell suspension was then pelleted and resuspended in DMEM containing 20%fetal calf serum and Antibiotic-Antimycotic (Invitrogen, Carlsbad,Calif.). Cells were cultured four days, and cryopreserved.

Plasmid Fragment Preparation for Transfection:

The pREV 859B plasmid fragment was prepared for transfection byrestriction enzyme digestion with BsmBI and AhdI. pREV 861 was preparedby digestion with BsmBI and EciI. pREV 872 was prepared by digestionwith DrdI. pREV 873 was prepared by digestion with BsmBI and EciI (allrestriction enzymes from New England Biolabs, Ipswitch, Mass.). Theplasmid fragments generated by digestion were separated on a 1% low meltagarose gel (Cambrex, East Rutherford, N.J.) to remove the plasmidbackbone. The transgene-containing cassette fragment of interest wasexcised and incubated twice in 2 volumes of 1X agarase buffer on ice for15 minutes. After removing the buffer, the gel was melted at 65° C. 10minutes. After 10 minutes at 42° C., 1 uL Agarase (New England Biolabs)per 100 uL of gel melt and incubated minimum 1 hour at 42° C. One-tenthvolume of 3M Sodium Acetate was added to the gel melt and incubated onice 15 minutes. Centrifugation at 15,000 rpm for 15 minutes at 4° C.separates any undigested agarose. Two volumes of 100% ethanol were addedto the supernatant and centrifugation was used to pellet the DNAfragment. 70% ethanol was used to wash the pellet before drying at 37°C. The pellet was resuspended in TE.

Transfection, Selection, Harvesting of Colonies for Screening:

Porcine fibroblasts from pig the 183-6-6 line were transfected witheither pREV872 (pICAM-2 hTM), or pREV859B (Tie-2 hCD39) and pREV828 (aPuromycin selectable marker gene vector)

pREV859B (Tie-2/hCD39) Transfection

Approximately 5 million cells were co-electroporated with 3 μg of thepREV859B vector and 0.5 μg of the selectable marker vector. Forty-eighthours post transfection, transfected cells were selected with theaddition of 1 μg/ml of the antibiotic Puromycin (InvivoGen, San Diego,Calif.) in 20×10 cm dishes at a density of approximately 25,000 cellsper dish. Media was changed 72 hours post initiation of puromycinselection. Colonies were harvested 9 days post initiation of selection.60 colonies grew and were split into two samples: one for PCR analysisand one for expansion. PCR analysis for pREV859B was performed asdescribed herein. Thirty-two PCR positive colonies were pooled andcryopreserved for future use in nuclear transfer.

pREV872 (ICAM2/hTM) Transfection

Approximately 5 million cells were co-electroporated with 5 μg of thepREV872 vector and 0.5 μg of the selectable marker vector. Forty-eighthours post transfection, transfected cells were selected with theaddition of 1 μg/ml of the antibiotic Puromycin (InvivoGen, San Diego,Calif.) in 30×10 cm dishes at a density of approximately 65,000 cellsper dish. Media was changed 72 hours post initiation of puromycinselection. Colonies were harvested 14 days post initiation of selection.22 colonies grew and were split into two samples: one for PCR analysisand one for expansion. PCR analysis for pREV872 was performed asdescribed herein. Three PCR positive colonies were pooled andcryopreserved for future use in nuclear transfer.

Similar procedures were used for transfection, selection and harvestingof colonies using the pREV861 vector and the pREV873 (pICAM-2 huEPCR)vector co-transfected in combination with the pREV872 (pICAM-2 huTM)vector.

Example 3

Production of Multi-Transgenic Pigs by Nuclear Transfer (NT)

Various methods can be used to produce the multi-transgenic pigs of thecurrent invention. The following is one example in which donor cellsused (line 227-3 and line 183-6-6) were the genetic backgroundhomozygous GTKO (lacked any function αGT) and were also transgenic forCD46. Donor cells were transfected, selected and screened positive forthe pREV859B, pREV861, pREV872, and/or pREV873 vectors, as described inExample 2, prior to being used for NT. In some cases, multiple coloniesof transfected and selected cells, all screening positive for thetransgene(s), were pooled together prior to their use in NT.

Donor cells (fetal or adult fibroblast cells) for NT were cultured inDulbecco's Modified Eagle Medium (DMEM, Gibco, cat #11995-065)supplemented with 10-20% fetal calf serum and 0-4 ng/ml basic fibroblastgrowth factor, in a humidified incubator at 5% O2, 5% CO2 balanced withnitrogen at 37° C. For culture, cells were seeded 3-7 days prior to thenuclear transfer procedure, at an appropriate dilution such that thecells would reach confluency 24-48 hours prior to nuclear transfer. Onthe day of nuclear transfer, donor cells were harvested about 30-45minutes before use in embryo reconstruction by using Trypsin-EDTA(Gibco, cat #25300-054), making a single cell suspension in suitableholding medium (e.g. Hepes buffered M199, Gibco cat #12350-039).

NT procedures were performed on in vitro matured oocytes (DesotoBiosciences, Christiansburg, Va.) using methods well known in the art(see, e.g., Polejaeva, et al., (2000) Nature 407, 86-90, Dai et al.,(2002) Nature biotechnology 20, 251-255, Campbell et al., (2007)Theriogenology 68 Suppl 1, S214-231, Vatja et al., (2007) Reprod FertilDev 19, 403-423). Electrical fusion and activation of reconstructedoocytes was performed using an ECM2001 Electrocell Manipulator (BTXInc., San Diego). Fused and activated nuclear transfer embryos were heldin culture in phosphate buffered NCSU-23 medium (J Rprod Fertil Suppl.1993; 48:61-73) for 1-4 h at 38.5° C., and then transferred to theoviduct of an estrus-synchronized recipient gilt. Crossbred gilts (largewhite/Duroc/Landrace) (280-400 lbs) were synchronized as recipientanimals by oral administration of 18-20 mg Matrix (Altrenogest, Hoechst,Warren, N.J.) mixed into their feed. Matrix was fed for 14 consecutivedays. Human Chorionic Gonadotropin (hCG, 1,000 units; Intervet America,Millsboro, Del.) was administered intramuscularly 105 h after the lastRegu-Mate treatment. Embryo transfers were performed by mid-ventrallaparotomy 22-26 h after the hCG injection. Pregnant Mare SerumGonadotropin (PMSG, 1,000 IU) and hCG (500 IU) we used on day 10 and 13post transfer for maintenance of pregnancy. Pregnancy was confirmed viaultrasonography 28 days post-transfer. Pregnancies were monitoredthereafter on a weekly basis. All piglets were born via naturalparturition.

Example 4

Genotyping of Cells and Transgenic Animals by PCR and Southern BlotAnalysis

Genotype Analysis:

Genomic DNA was extracted from transfected cells, and blood or tissuesamples from each piglet to be tested. In brief, tissue samples werelysed overnight at 60° C. in a shaking incubator with approximately 1 mllysis solution (50 mM Tris pH8.0, 0.15 M NaCl, 0.01 M EDTA, 1% SDS, 25%Sodium perchlorate and 1% of β-Mercaptoethanol and Proteinase K) per 175mg tissue. DNA was precipitated with isopropyl alcohol followingphenol/chloroform extraction. Resolubilized DNA was treated with RNase(1 mg/ml)+RNase T1 (1,000 U/μl) at 37° C. for 1 hour, with proteinase K(20 mg/ml) at 55° C. for 1 hour, extracted with phenol/chloroform,precipitated and resuspended in Tris Ethylenediaminetetraacetic acid(EDTA). DNA was isolated from whole blood samples using a DNA isolationkit for mammalian blood (Roche Diagnostics Indianapolis, Ind.).

For Southern blot analysis, about 10 μg of DNA was digested with theappropriate restriction enzyme (detail below) and separated on a 1%agarose gel. Following electrophoresis, the DNA was transferred to anylon membrane and probed with a 3′-end digoxigenin-labeled probe (probesequence below). Bands were detected using a chemiluminescent substratesystem (Roche Diagnostics, Indianapolis, Ind.).

Primers and Probes:

pREV859B—Tie-2/huCD39

The presence of integrated pREV859B construct was determined by PCRusing primers CD39L3 and CD39R3 which target a 585 bp fragment withinthe CD39 coding sequence.

CD39L3: AGTATGGGATTGTGCTGGATG CD39R3: CATAGAGGCGAAATTGCAGAG

The presence of integrated pREV859B construct was confirmed by Southernblot analysis using a BanHI digest and probing with probe CD39L3/R3-dig.

CD39L3/R3-Dig Probe Sequence:

AGTATGGGATTGTGCTGGATGCGGGTTCTTCTCACACAAGTTTATACATCTATAAGTGGCCAGCAGAAAAGGAGAATGACACAGGCGTGGTGCATCAAGTAGAAGAATGCAGGGTTAAAGGTCCTGGAATCTCAAAATTTGTTCAGAAAGTAAATGAAATAGGCATTTACCTGACTGATTGCATGGAAAGAGCTAGGGAAGTGATTCCAAGGTCCCAGCACCAAGAGACACCCGTTTACCTGGGAGCCACGGCAGGCATGCGGTTGCTCAGGATGGAAAGTGAAGAGTTGGCAGACAGGGTTCTGGATGTGGTGGAGAGGAGCCTCAGCAACTACCCCTTTGACTTCCAGGGTGCCAGGATCATTACTGGCCAAGAGGAAGGTGCCTATGGCTGGATTACTATCAACTATCTGCTGGGCAAATTCAGTCAGAAAACAAGGTGGTTCAGCATAGTCCCATATGAAACCAATAATCAGGAAACCTTTGGAGCTTTGGACCTTGGGGGAGCCTCTACACAAGTCACTTTTGTACCCCAAAACCAGACTATCGAGTCCCCAGATAATGCTCTGCAATTTCGCCTCTATG

In some cases, probe Tie2L/R-dig was used:

TGGCAGCTTCTGCTTGCTTCGATCAGCTGCCAGTTAGGTAGCAACAAACTTGGGATAAGTAACATAAGGAGGGTAGTTACAAGCAACAAGTCATCTTAGAACCTCTGCTAAGTCAAGACCCAGAGGCAAGAAGAAGTTGGGAATTGGTTGGGGAAAAGTAGGGGGCTCCACCTTGCTGGCTGGCTGAGTCACAAGCAAGGAATTTCCCCACCAGACAACCCAGCTTTTTAACAGAAGCCCAGGAACGCAAAGCTTTAAGCCCTTCTCTTCGTTTTCCTGATACAAAGATGCTCTTTTGCAGTCAAAGCAGCCAGAGTCAGCCCCACACATATATAAACAGATTAGCTCAGGAATGGAGGCCTGCCC TGAApREV861—pICAM2/CD39

The presence of integrated pREV861 construct was determined by PCR usingprimers CD39L3 and CD39R3 which targets a 585 bp fragment within theCD39 coding region.

CD39L3: AGTATGGGATTGTGCTGGATG CD39R3: CATAGAGGCGAAATTGCAGAG

The presence of integrated pREV861 construct was confirmed by Southernblot analysis using a BamHI digest and probing with probe CD39L3/R3-dig.

CD39L3/R3-Dig Probe Sequence:

AGTATGGGATTGTGCTGGATGCGGGTTCTTCTCACACAAGTTTATACATCTATAAGTGGCCAGCAGAAAAGGAGAATGACACAGGCGTGGTGCATCAAGTAGAAGAATGCAGGGTTAAAGGTCCTGGAATCTCAAAATTTGTTCAGAAAGTAAATGAAATAGGCATTTACCTGACTGATTGCATGGAAAGAGCTAGGGAAGTGATTCCAAGGTCCCAGCACCAAGAGACACCCGTTTACCTGGGAGCCACGGCAGGCATGCGGTTGCTCAGGATGGAAAGTGAAGAGTTGGCAGACAGGGTTCTGGATGTGGTGGAGAGGAGCCTCAGCAACTACCCCTTTGACTTCCAGGGTGCCAGGATCATTACTGGCCAAGAGGAAGGTGCCTATGGCTGGATTACTATCAACTATCTGCTGGGCAAATTCAGTCAGAAAACAAGGTGGTTCAGCATAGTCCCATATGAAACCAATAATCAGGAAACCTTTGGAGCTTTGGACCTTGGGGGAGCCTCTACACAAGTCACTTTTGTACCCCAAAACCAGACTATCGAGTCCCCAGATAATGCTCTGCAATTTCGCCTCTATG

pREV872—pICAM2/huTM

The presence of integrated pREV872 construct was determined by PCR usingprimers TML and TMR which targets a 533 by fragment within the TM codingregion.

TML: ACTGCAGCGTGGAGAACGGC TMR: GGTGTTGGGGTCGCAGTCGG

The presence of integrated pREV872 construct was confirmed by Southernblot analysis using a BamHI digest and probing with probe TML/R-dig.

TML/R-dig probe sequence:

ACTGCAGCGTGGAGAACGGCGGCTGCGAGCACGCGTGCAATGCGATCCCTGGGGCTCCCCGCTGCCAGTGCCCAGCCGGCGCCGCCCTGCAGGCAGACGGGCGCTCCTGCACCGCATCCGCGACGCAGTCCTGCAACGACCTCTGCGAGCACTTCTGCGTTCCCAACCCCGACCAGCCGGGCTCCTACTCGTGCATGTGCGAGACCGGCTACCGGCTGGCGGCCGACCAACACCGGTGCGAGGACGTGGATGACTGCATACTGGAGCCCAGTCCGTGTCCGCAGCGCTGTGTCAACACACAGGGTGGCTTCGAGTGCCACTGCTACCCTAACTACGACCTGGTGGACGGCGAGTGTGTGGAGCCCGTGGACCCGTGCTTCAGAGCCAACTGCGAGTACCAGTGCCAGCCCCTGAACCAAACTAGCTACCTCTGCGTCTGCGCCGAGGGCTTCGCGCCCATTCCCCACGAGCCGCACAGGTGCCAGATGTTTTGCAACCAGACTGCCTGTCCAGCCGACTGCGACCCCAACACCpREV873—pICAM2/huEPCR

The presence of integrated pREV873 construct was determined by PCR usingprimers EPCR5′ and 858R3381 which targets a 692 by fragment from withinthe huEPCR coding region to outside of the huEPCR coding region

EPCR5′: TCCTGGGCTGTGAGCTGCCT 858.R3381: CCCCCTGAACCTGAAACATA

The presence of integrated pREV873 construct was confirmed by Southernblot analysis using a BamHI digest and probing with EPCR5′/3′ dig probe.

EPCR3′/3′ Dig Probe Sequence:

TCCTGGGCTGTGAGCTGCCTCCCGAGGGCTCTAGAGCCCATGTCTTCTTCGAAGTGGCTGTGAATGGGAGCTCCTTTGTGAGTTTCCGGCCGGAGAGAGCCTTGTGGCAGGCAGACACCCAGGTCACCTCCGGAGTGGTCACCTTCACCCTGCAGCAGCTCAATGCCTACAACCGCACTCGGTATGAACTGCGGGAATTCCTGGAGGACACCTGTGTGCAGTATGTGCAGAAACATATTTCCGCGGAAAACACGAAAGGGAGCCAAACAAGCCG CTCCTACACTTCGCTGGTCCTGGGCG

Example 5

Phenotypic Analysis (pCTLA4-Ig) of Tissues from Transgenic Pigs

Western Blot for pCTLA4-Ig Expression:

Tissue and cell lysates can be prepared by homogenization in thepresence of protease inhibitors (Thermo Scientific, Rockford, Ill.)followed by the addition of SDS (1% final concentration) andcentrifugation to remove residual cellular debris. Protein concentrationis determined with a bicinchoninic acid (BCA) protein assay kit (Pierce,Rockford, Ill.). Heat-denatured and β-mercaptoethanol-reduced samples(10-20˜g protein) are fractionated on 4-12% BisTris SDS gradient gels(Invitrogen, Carlsbad, Calif.). Recombinant human CTLA4-Ig/Fc (R&DSystems, Minneapolis, Minn.) is used as a standard control protein.Following electrophoresis, proteins are transferred to a nitrocellulosemembrane, stained with Memcode Protein Stain (Thermo Scientific) fortotal protein visualization, and blocked with casein-blocking buffer(Sigma-Aldrich., St. Louis, Mo.). The blocked membrane is incubated inrabbit anti-human IgG1-horseradish peroxidase (HRP) (The Binding Site,San Diego, Calif.), which recognizes the human IgG1 heavy chain regionof pCTLA4-Ig. Immunoreactive bands are detected with Super Signal WestPico Chemiluminescent Substrate (Thermo Scientific) and photographicimaging.

Example 6

Phenotypic Analysis of Animals Expressing Transgenes in Endothelium

In order to screen for expression of the various transgenes in theendothelium of transgenic pigs produced, aortic endothelial cells wereprocured from animals determined to be genotypically positive andexamined via flow cytometry.

Aortic Endothelial Cell Isolation

3 to 6 inches of descending aorta (vessel) were removed from theeuthanatized pig and placed in RPMI+Antibiotic/Antimycotic (Invitrogen,Carlsbad, Calif.). The vessel was thoroughly washed using DBPS(Mediatech, Inc. Manassas, Va.) by flushing the interior and exterior ofthe vessel with multiple volumes of DPBS to remove blood. The exteriorof the vessel was trimmed of any excess tissue such as muscle and fat.Both ends of vessel were clamped closed. The vessel was filled withRPMI+100 units of activity/ml of collagenase type 4 (WorthingtonBiochemical, Lakewood, N.J.). It was then incubated for 15-30 min at 37°C. The clamps were removed and the vessel contents were emptied into a15 ml tube and the vessel was flushed with an additional 10 mls of RPMI.This collagenase digestion was repeated up to three times. The fractionswere kept separate. The cell fractions were pelleted and washed with 10mls of RPMI. Each cell fraction was seeded in separate 10 cm plates in10 mls of RPMI+10% FBS+1× Antibiotic/antimycotic.

Detection of Anticoagulant Transgene Expression (TM and CD39) onEndothelial Cells Via Flow Cytometry

Endothelial cells were harvested 24-72 hr post isolation to measureexpression of TM or CD39 by flow cytometry. The number of endothelialcells was counted and adjusted by dilution or concentration to 10×10⁶per ml in Stain buffer (BD Pharmingen, San Diego, Calif.). The cellswere exposed to antibody as per manufacturer's suggestion at aconcentration of 20 μl of antibody per 1.0×10⁶ cells.

For TM expression: PE labeled Anti-human CD141 (BD Pharmingen, SanDiego, Calif.) was used. PE mouse IgG1 k was used as the isotypecontrol.

For CD39 expression: PE labeled Anti-human CD39 (BD Pharmingen, SanDiego, Calif.) was used. PE mouse IgG2b was used as the isotype control.

Cells were incubated with the appropriate antibody for 30 min at 4° C.Cells were then washed with 2-5 ml of stain buffer. Cells wereresuspended in 0.5 ml of Stain buffer. Antibody labeling was recorded bymeasuring PE expression level of 10,000 cells per sample using flowcytometry.

Histology and Immunofluorescence (IF):

Porcine endothelial tissue samples can be removed and either fixed in10% formalin or frozen down in blocks of OCT (Electron MicroscopySciences, Hatfield, Pa.). Frozen sections are cut at 5 μm on a cryostatand are stained with rabbit anti-human TFPI (polyclonal, AmericanDiagnostica, Stamford, Conn., #4901), sheep anti-human IgG1 (polyclonal,The Binding Site, Birmingham, UK, #AUOO6), mouse anti-human CD46 (cloneO.N. 137, mIgG2a, U.S. Biological, Swampscott, Mass.), mouse anti-humanCD39 ((clone A1, AbD Serotec, Oxford, UK), mouse anti-human CD201(EPCR)(clone RCR-252, BD Pharmingen, San Jose, Calif.) or mouse anti-humanCD141(TM) (clone 1A4, BD Pharmingen). Isotype controls are run forrabbit IgG (Jackson ImmunoResearch, West Grove, Pa.), sheep IgG(Jackson), mouse IgG2a (BD Pharmingen) and mouse IgG1 (clone MOPC-31C,BD Pharmingen), respectively. Immunofluorescent (IF) staining isperformed using a 3-step procedure. Frozen sections are dried and fixedin cold acetone (Sigma, St. Louis, Mo.), followed by avidin-biotinblocking (Invitrogen, Carlsbad, Calif.). Secondary Ab host species serumblocking steps are also included (10% Donkey serum, Jackson). PrimaryAbs are diluted in PBS and incubations are performed for 1 h at roomtemperature in a humidified chamber. The secondary Ab used isbiotinylated donkey anti-(rabbit, sheep, or mouse) IgG for 45 mm and thetertiary Ab used is fluorescein-conjugated strep avidin for 30 mm(Jackson). Slides are washed in PBS between steps, are cover slippedusing 22×30 mm coverslips (VWR, West Chester, Pa.) and are preservedusing Slowfade with DAPI (Invitrogen). IF pictures can be taken using anOlympus DP71 camera on a Provis microscope, and analyzed using DPcontroller software (Olympus, Center Valley, Pa.) with a magnificationof 200×.

Cell Smears for IF Analysis:

In some cases cell-smears can be prepared from organs and tissuescontaining endothelium, to determine presence of the transgenic proteinvia IF. The following procedure is followed:

Approximately 1×1 cm of tissue is placed in a 4 ml snap cap tube and 1ml of DMEM+collagenase at 50-100 units activity/ml is added. The tube isincubated for 10 min at 37° C. Next the tissue is minced using a longhandle scissor by placing the scissors in the tube and opening andclosing scissor blades for 3-5 min. The tube is then incubated for 10min at 37° C. Mincing is repeated for 3-5 more minutes, 2 ml of DPBS isadded and the resulting cells are pelleted via centrifugation. They arethen washed in 3 ml DPBS and resuspended in 250 μl of Cytofix FixationBuffer (BD Biosciences). They are incubated in the buffer for 20 min at4° C. Next, 2 ml of DPBS is added and the cells are pelleted. They arewashed in 3 ml of distilled water and then resuspended in 1 ml ofdistilled water. 5 μl of cell solution is placed on a superfrost plusglass slide. Slides are allowed to air dry and can be stored at 4° C.for up to one week. Slides are stained following the same IF protocol asfor blocked and sectioned tissues (see above).

Real Time PCR (RTPCR) to Measure TM Transcript in Samples fromMulti-Transgenic Pigs

Lung, liver, heart, aorta and kidney samples were obtained from piglets448-01, 448-02, 448-03 and 450-06 postmortem. Tissues were homogenizedand total RNA was isolated using Trizol (Invitrogen, Carlsbad, Calif.)following the procedure of Chomcyznski and Sacchi (Anal Biochem. 1987April; 162(1):156-9). Reverse transcription was performed using theiScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Hercules,Calif.) according to the manufacturer's instructions. A reaction mixcontaining 1 μg of RNA was formulated for the sample, a non-reversetranscriptase and a non-template control reaction. In addition, all thesamples were treated with DNase I (Invitrogen, Carlsbad, Calif.) toprevent DNA contamination.

PCR primers for the amplification of hTM were designed from the 872construct sequence (forward primer: TTCAGAGCCAACTGCGAGTA and reverseprimer: AACCGTCGTCCAGGATGTAG). cDNA was amplified using iQTMSYBR GreenSupermix in the MyiQ Reverse Transcription PCR Detection System (Bio-RadLaboratories, Inc., Hercules, Calif., USA). Complementary DNA wasamplified using SYBR Green PCR Master Mix in the ABI Prism® 7,000Sequence Detection System (Applied Biosystems, Foster City, Calif.). Ano reverse transcriptase, a wild type and a no template sample wereincluded in every plate as negative controls. Three replicates of everytissue were analyzed. The copy number of hTM in all the tissues wascalculated using the standard curve method.

Results

Multitransgenic Pigs Produced, Genotypic and Phenotypic Characterization

Five sessions of nuclear transfer, using 183-6-6 cells screenedtransgenic for the pREV859B, pREV861, pREV872 or pREV873 anticoagulanttransgenes, as nuclear donors, resulted in the production of fivelitters of piglets. Thirty three piglets were born, and 23 were aliveafter birth. Fourteen of these piglets screened positive for ananticoagulant transgene in their genome (thirteen transgenic for TM, andone transgenic for CD39). In some cases, two different anticoagulanttransgenes (TM and EPCR) were present in the same piglet. The CD39multi-transgenic piglet was shown to express CD39 in endothelium via IFflow cytometry of isolated endothelial cells. The thirteenmulti-transgenic TM pigs were all shown to express TM in endothelium viaIF flow cytometry of isolated endothelial cells. Additionally, a subsetof these multi-transgenic TM piglets were tested viaimmunohistochemistry (IHC) and showed IF expression of TM in organs (viacell smear) and/or endothelium of tail tissue (FIG. 4). Transcriptexpression of huTM via RTPCR was also determined (FIG. 5). The tablebelow details genotype and TM protein expression data in these animals.

TABLE 1 Multi-transgenic pigs* produced with endothelial-specificanticoagulant transgenes. Piglet Generation and Genotype Phenotype DataVector present in Flow transgenic cell(s) Cytometry used to generatePiglet Anticoagulant (Endo), Organ Cell piglets via NT ID Genotype TMIHC, TM Smear, TM pREV872, pREV873 424-01 TM/EPCR (+) (+) tail (+) ht,ki, li, lu pREV872, pREV873 424-02 TM/EPCR (+) (+) lu, li, ht, ao, ki ndpREV872, pREV873 424-03 TM/EPCR (+) (+) tail nd pREV872 448-01 TM (+) nd(+) ht, ki, li, lu pREV872 448-02 TM (+) nd (+) ht, ki, li, lu pREV872448-03 TM (+) (+) lu, li, ht, ao (+) ht, ki, li, lu pREV872 448-04 TM(+) nd (+) ht, ki, li, lu pREV872 448-05 TM (+) nd (+) ht, ki, li, lupREV872 450-01 TM (+) nd nd pREV872 450-05 TM (+) nd nd pREV872 450-06TM (+) (+)lu, li, ht, ao (+) ht, ki, li, lu pREV872 450-07 TM (+) nd ndpREV872 451-03 TM (+) nd (+) ht, ki, li, lu Flow Cytometry DonorTransgenic Piglet Anticoagulant (Endo), Organ Cell Cell(s) (used for NT)ID Genotype CD39 IHC, CD39 Smear, CD39 pREV859B, pREV861 440-04 (Tie-2)CD39 (+) (+) ht, ki, li, lu, ao nd *All pigs were additionally trangenicfor the GTKO genetic modification and the CD46 transgene. (Data notshown). This is the background genetics of the 183-6-6 donor cell lineused to generate the multi-transgenic piglets with endothelial specifictransgenes.

FIG. 3 shows TM expression in endothelial cells isolated from piglet424-01 determined via flow cytometry and CD39 expression in endothelialcells isolated from piglet 440-04. Samples of tail and organ tissuescontaining endothelium were collected from piglet 424-01 atapproximately one month of age and phenotypically characterized forendothelial expression of TM by IF as described in Example 6.

FIG. 4 shows endothelial specific expression of TM determined via IHC oftail tissue from piglet 424-03.

FIG. 5 shows TM transcript expression by RTPCR in samples obtained frommulti-transgenic piglets 448-01, 448-02, 448-03 and 450-06. TM copynumber shown is the copy number of hTM present in 50 ng of cDNA.

The invention claimed is:
 1. A transgenic porcine animal comprisinggenetic modifications that result in: (i) the lack of expression offunctional alpha 1,3 galactosyltransferase; (ii) incorporation into thegenome and expression of (a) a complement inhibitor transgene, whereinthe complement inhibitor transgene is CD46, wherein the complementinhibitor transgene is ubiquitously expressed and is under the controlof a constitutive promoter; (b) one immunosuppressant transgene underthe control of a constitutive promoter, wherein the oneimmunosuppressant transgene is selected from the group consisting ofCytotoxic T-Lymphocyte-Associated Protein 4 (CTLA4), cluster ofdifferentiation 47 (CD47) and Class II transactivator-DN (CIITA-DN); and(c) two anticoagulant transgenes under the control of anendothelial-specific promoter, wherein the two anticoagulant transgenesare endothelial protein C receptor (EPCR) and thrombomodulin.
 2. Cellsderived from the transgenic porcine animal of claim
 1. 3. An organderived from the transgenic porcine animal of claim
 1. 4. The organ ofclaim 3, wherein the organ is selected from the group consisting ofheart, lung, liver and kidney.
 5. Tissue derived from the transgenicporcine animal of claim
 1. 6. The tissue of claim 5, wherein the tissueis selected from the group consisting of vascular tissue, heart valve,retinal tissue and corneal tissue.
 7. The tissue of claim 6, wherein thevascular tissue is a vascular graft.
 8. The transgenic animal of claim1, wherein the immunosuppressant is CD47.
 9. The transgenic animal ofclaim 1, wherein the immunosuppressant is CIITA-DN.
 10. A transgenicporcine animal comprising genetic modifications that result in: (i) thelack of expression of functional alpha 1,3 galactosyltransferase; and(ii) incorporation into the genome and expression of (a) at least onecomplement inhibitor transgene under the control of a constitutivepromoter, and wherein the complement inhibitor transgene is CD46 and isubiquitously expressed; (b) at least one immunosuppressant transgeneexpressed under the control of a constitutive promoter, wherein the atleast one immunosuppressant transgene is CD47 or CTIIA-DN; and (c) atleast two anticoagulant transgenes under the control of anendothelial-specific promoter, wherein the anticoagulant transgenes areendothelial protein C receptor (EPCR) and thrombomodulin.
 11. Cellsderived from the transgenic porcine animal of claim
 10. 12. An organderived from the transgenic porcine animal of claim
 10. 13. Tissuederived from the transgenic porcine animal of claim
 10. 14. The animalof claim 1 or 10 wherein the endothelial-specific promoter is ICAM-2 orTie-2.
 15. A method for xenotransplantation comprising administering, toa primate in need thereof, porcine organs, tissue or cells derived fromthe transgenic animal of claim
 1. 16. The method of claim 15, whereinthe primate is a non-human primate.
 17. The method of claim 16, whereinthe primate is a human.
 18. The method of claim 15, wherein the organ isselected from the group consisting of heart, lung, liver and kidney. 19.The method of claim 15, wherein the tissue is selected from the groupconsisting of vascular tissue, retinal tissue and corneal tissue. 20.The method of claim 15, further comprising administering a clinicallyrelevant immunosuppressant regimen to the primate followingxenotransplantation of the organs, tissues or cells.
 21. The method ofclaim 20 wherein the primate is human.